U.S. patent application number 12/303088 was filed with the patent office on 2011-02-03 for lyase enzymes, nucleic acids encoding them and methods for making and using them.
This patent application is currently assigned to VERENIUM CORPORATION. Invention is credited to Ellen Burke, Shaun Healey, Mircea Podar, Toby Richardson, Alexander Varvak, David Weiner.
Application Number | 20110027346 12/303088 |
Document ID | / |
Family ID | 39789151 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110027346 |
Kind Code |
A1 |
Weiner; David ; et
al. |
February 3, 2011 |
Lyase Enzymes, Nucleic Acids Encoding Them and Methods for Making
and Using Them
Abstract
This invention provides polypeptides having lyase activity,
polynucleotides encoding these polypeptides, and methods of making
and using these polynucleotides and polypeptides. In one aspect,
the invention is directed to polypeptides having ammonia lyase
activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase activity, including thermostable and
thermotolerant activity, and polynucleotides encoding these
enzymes, and making and using these polynucleotides and
polypeptides. The polypeptides of the invention can be used in a
variety of pharmaceutical, agricultural and industrial
contexts.
Inventors: |
Weiner; David; (Del Mar,
CA) ; Varvak; Alexander; (San Diego, CA) ;
Richardson; Toby; (San Diego, CA) ; Podar;
Mircea; (San Diego, CA) ; Burke; Ellen; (San
Diego, CA) ; Healey; Shaun; (Carlsbad, CA) |
Correspondence
Address: |
VERENIUM CORPORATION;Intellectual Property Department
P.O. Box 910550
SAN DIEGO
CA
92191-0550
US
|
Assignee: |
VERENIUM CORPORATION
San Diego
CA
|
Family ID: |
39789151 |
Appl. No.: |
12/303088 |
Filed: |
May 29, 2007 |
PCT Filed: |
May 29, 2007 |
PCT NO: |
PCT/US07/69877 |
371 Date: |
September 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60810528 |
Jun 2, 2006 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/484; 424/94.5; 426/42; 435/136; 435/176; 435/180; 435/232;
435/252.3; 435/254.11; 435/254.2; 435/268; 435/320.1; 435/325;
435/348; 435/419; 506/18; 536/23.2; 536/24.3; 977/906 |
Current CPC
Class: |
A61P 7/00 20180101; C12N
9/88 20130101 |
Class at
Publication: |
424/450 ;
536/23.2; 536/24.3; 435/320.1; 435/252.3; 435/325; 435/254.11;
435/254.2; 435/348; 435/419; 435/232; 435/176; 435/180; 506/18;
435/136; 424/94.5; 424/484; 435/268; 426/42; 977/906 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 1/21 20060101 C12N001/21; C12N 5/10 20060101
C12N005/10; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101
C12N001/19; A61P 7/00 20060101 A61P007/00; C12N 9/88 20060101
C12N009/88; C12N 11/16 20060101 C12N011/16; C12N 11/14 20060101
C12N011/14; C12N 11/08 20060101 C12N011/08; C40B 40/10 20060101
C40B040/10; C12P 7/40 20060101 C12P007/40; A61K 38/51 20060101
A61K038/51; A61K 9/14 20060101 A61K009/14; A23C 9/12 20060101
A23C009/12 |
Claims
1. An isolated, synthetic, or recombinant nucleic acid comprising:
(a) a nucleic acid sequence having at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more or complete sequence identity to SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ
ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,
SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,
SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID
NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,
SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139,
SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID
NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,
SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID
NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,
SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID
NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193,
SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID
NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211,
SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID
NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,
SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID
NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247,
SEQ ID NO:249 or SEQ ID NO:101, or a fragment thereof, wherein the
nucleic acid encodes at least one polypeptide having a lyase
activity; or (b) a nucleic acid sequence that hybridizes under
stringent conditions to the complement of a nucleic acid comprising
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ
ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37,
SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID
NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ
ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65,
SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ
ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,
SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99 or SEQ ID NO:101, wherein
the nucleic acid encodes a polypeptide having a lyase activity, and
the stringent conditions include a wash step comprising a wash in
0.2.times.SSC at a temperature of about 65.degree. C. for about 15
minutes; (c) a nucleic acid sequence encoding a polypeptide having
a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ
ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100 or SEQ ID NO:102; or (d) a nucleic acid sequence
complementary to (a), (b) or (c).
2. The isolated or recombinant nucleic acid of claim 1, wherein the
nucleic acid sequence comprises a sequence as set forth in SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ
ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,
SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99 or SEQ ID NO:101.
3. (canceled)
4. The isolated or recombinant nucleic acid of claim 1, wherein the
lyase activity comprises an ammonia lyase activity.
5-30. (canceled)
31. A nucleic acid probe for identifying a nucleic acid encoding a
polypeptide with a lyase activity, wherein the probe comprises at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100,
125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more
consecutive bases of a sequence as set forth in claim 1, wherein
the probe identifies the nucleic acid by binding or
hybridization.
32-36. (canceled)
37. An expression cassette, a vector, a transformed cell, or a
cloning vehicle comprising the nucleic acid of claim 1.
38. (canceled)
39. The cloning vehicle of claim 37 comprising: a viral vector, a
plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage,
or an artificial chromosome, wherein optionally the viral vector
comprises an adenovirus vector, a retroviral vector or an
adeno-associated viral vector, and optionally the cloning vehicle
comprises a bacterial artificial chromosome (BAC), a plasmid, a
bacteriophage P1-derived vector (PAC), a yeast artificial
chromosome (YAC), or a mammalian artificial chromosome (MAC).
40. The transformed cell of claim 37 wherein the transformed cell
is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell,
an insect cell, or a plant cell.
41-47. (canceled)
48. An isolated, synthetic, or recombinant polypeptide: (i) having
lyase activity and an amino acid sequence having at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,
SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID
NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ
ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76,
SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ
ID NO:96, SEQ ID NO:98, SEQ ID NO:100 or SEQ ID NO:102, or a
fragment thereof, wherein the polypeptide has a lyase activity;
(ii) an amino acid sequence encoded by a nucleic acid as set forth
in claim 1, wherein the polypeptide has a lyase activity; or (iii)
an amino acid sequence as set forth in (i) or (ii), and comprising
at least one amino acid residue conservative substitution and
retaining its lyase activity or immunogenic activity, wherein
optionally conservative substitution comprises replacement of an
aliphatic amino acid with another aliphatic amino acid; replacement
of a serine with a threonine or vice versa; replacement of an
acidic residue with another acidic residue; replacement of a
residue bearing an amide group with another residue bearing an
amide group; exchange of a basic residue with another basic
residue; or, replacement of an aromatic residue with another
aromatic residue, or a combination thereof, and optionally the
aliphatic residue comprises Alanine, Valine, Leucine, Isoleucine or
a synthetic equivalent thereof; the acidic residue comprises
Aspartic acid, Glutamic acid or a synthetic equivalent thereof the
residue comprising an amide group comprises Aspartic acid, Glutamic
acid or a synthetic equivalent thereof the basic residue comprises
Lysine, Arginine or a synthetic equivalent thereof or, the aromatic
residue comprises Phenylalanine, Tyrosine or a synthetic equivalent
thereof.
49. The isolated or recombinant polypeptide of claim 48, wherein
the lyase activity comprises an ammonia lyase activity.
50-75. (canceled)
76. The isolated or recombinant polypeptide comprising a
polypeptide as set forth in claim 48 and lacking a signal or leader
sequence or a prepro sequence.
77. An isolated or recombinant polypeptide comprising a polypeptide
as set forth in claim 48 and having a heterologous signal or leader
sequence or a heterologous prepro sequence.
78. (canceled)
79. (canceled)
80. The isolated or recombinant polypeptide of claim 48, wherein
the polypeptide comprises at least one glycosylation site, and
optionally the glycosylation is an N-linked glycosylation, and
optionally the polypeptide is glycosylated after being expressed in
a yeast cell or mammalian cell, and optionally the yeast cell is P.
pastoris or a S. pombe.
81. (canceled)
82. (canceled)
83. A protein preparation comprising a polypeptide as set forth in
claim 48, wherein the protein preparation comprises a liquid, a
solid or a gel.
84. A heterodimer comprising a polypeptide as set forth in claim 48
and a second domain, wherein optionally the second domain is a
polypeptide and the heterodimer is a fusion protein, and optionally
the second domain comprises an epitope, an immunogenic peptide or a
tag.
85. A homodimer comprising a polypeptide as set forth in claim
48.
86. An immobilized polypeptide or an immobilized nucleic acid,
wherein the polypeptide comprises a sequence as set forth in claim
48, or a subsequence thereof, or the nucleic acid comprises a
sequence as set forth in claim 1, or a subsequence thereof, or the
probe as set forth in claim 31, wherein optionally the polypeptide
or nucleic acid is immobilized on a cell, a metal, a resin, a
polymer, a ceramic, a glass, a microelectrode, a graphitic
particle, a bead, a gel, a plate, an array or a capillary tube.
87-91. (canceled)
92. A method of producing a recombinant polypeptide comprising the
steps of: (a) providing the nucleic acid of claim 1; and (b)
expressing the nucleic acid of step (a) under conditions that allow
expression of the polypeptide, thereby producing a recombinant
polypeptide. wherein optionally the method further comprises
transforming a host cell with the nucleic acid of step (a) followed
by expressing the nucleic acid of step (a), thereby producing a
recombinant polypeptide in a transformed cell.
93-105. (canceled)
106. A method of generating a variant of a nucleic acid encoding a
polypeptide with a lyase activity comprising the steps of: (a)
providing a template nucleic acid comprising a sequence as set
forth in claim 1; and (b) modifying, deleting or adding one or more
nucleotides in the template sequence, or a combination thereof, to
generate a variant of the template nucleic acid wherein optionally
the method further comprises expressing the variant nucleic acid to
generate a variant lyase polypeptide, and optionally the
modifications, additions or deletions are introduced by a method
comprising error-prone PCR, shuffling, oligonucleotide-directed
mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo
mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis,
exponential ensemble mutagenesis, site-specific mutagenesis, gene
reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic
ligation reassembly (SLR), recombination, recursive sequence
recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis,
point mismatch repair mutagenesis, repair-deficient host strain
mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion
mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, artificial gene synthesis,
ensemble mutagenesis, chimeric nucleic acid multimer creation and a
combination thereof and optionally the method is iteratively
repeated until a lyase having an altered or different activity or
an altered or different stability from that of a polypeptide
encoded by the template nucleic acid is produced.
107-109. (canceled)
110. A method for modifying codons in a nucleic acid encoding a
lyase polypeptide, the method comprising the following steps: (a)
providing a nucleic acid encoding a polypeptide with a lyase
activity comprising a sequence as set forth in claim 1; and, (b)
identifying a codon in the nucleic acid of step (a) and replacing
it with a different codon encoding the same amino acid as the
replaced codon, thereby modifying codons in a nucleic acid encoding
a lyase.
111-127. (canceled)
128. A method for deaminating a phenylalanine, tyrosine or a
histidine comprising the following steps: (a) providing a
polypeptide having a lyase activity as set forth in claim 48, or a
polypeptide encoded by a nucleic acid as set forth in claim 1; (b)
providing a composition comprising a phenylalanine, tyrosine or a
histidine residue; and (c) contacting the polypeptide of step (a)
with the composition of step (b) under conditions wherein the lyase
deaminates the phenylalanine, tyrosine or a histidine residue in
the composition. wherein optionally the composition comprises a
plant cell, a bacterial cell, a yeast cell, an insect cell, or an
animal cell, and optionally the polypeptide has ammonia lyase
activity.
129-131. (canceled)
132. A beverage, a drink, a food, a feed or a nutritional
supplement comprising a polypeptide as set forth in claim 48.
133-136. (canceled)
137. A wood, wood pulp, wood product, paper, paper pulp, paper
product, textile, or fabric comprising a lyase as set forth in
claim 48.
138. (canceled)
139. A detergent composition comprising a lyase as set forth in
claim 48.
140. A pharmaceutical composition or dietary supplement comprising
a lyase as set forth in claim 48.
141. The pharmaceutical composition or dietary supplement of claim
140, formulated for the treatment of phenylketonuria (PKU).
142. The pharmaceutical composition or dietary supplement of claim
140, wherein the polypeptide is chemically modified.
143. The pharmaceutical composition or dietary supplement of claim
142, wherein the polypeptide is chemically modified to produce a
protected form that possesses better specific activity, prolonged
half-life, and/or reduced immunogenicity in vivo.
144. (canceled)
145. The pharmaceutical composition or dietary supplement of claim
140, wherein the polypeptide is formulated by encapsulation in a
liposome, or a micro- or nano-structure, wherein optionally the
structure is a nanotubule or a nano- or microcapsule.
146. The pharmaceutical composition or dietary supplement of claim
140, wherein the polypeptide is formulated in a matrix stabilized
enzyme crystal.
147. A method for decreasing elevated levels of phenylalanine (Phe)
in the bloodstream (hyperphenylalaninemia) comprising the following
steps: (a) providing a pharmaceutical composition or dietary
supplement of any of claims 140 to 143; and, (b) administering an
effective amount of the pharmaceutical composition or dietary
supplement to an individual in need thereof.
148. A method for processing a biomass material comprising
contacting a composition with a polypeptide as set forth in claim
48.
149. (canceled)
150. A method for improving texture and flavor of a dairy product
comprising the following steps: (a) providing a polypeptide as set
forth in claim 48; (b) providing a dairy product; and (c)
contacting the polypeptide of step (a) and the dairy product of
step (b) under conditions wherein the lyase can improve the texture
or flavor of the dairy product.
151. (canceled)
152. A method for treating solid or liquid animal waste products
comprising the following steps: (a) providing a polypeptide as set
forth in claim 48; (b) providing a solid or a liquid animal waste;
and (c) contacting the polypeptide of step (a) and the solid or
liquid waste of step (b) under conditions wherein the protease can
treat the waste.
153-154. (canceled)
155. A biodefense or bio-detoxifying agent comprising a polypeptide
having a lyase activity, wherein the polypeptide comprises a
sequence as set forth in claim 48.
156-158. (canceled)
159. A composition comprising the polypeptide of claim 48 or the
nucleic acid of claim 1.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0001] This application is being filed electronically via the USPTO
EFS-WEB server, as authorized and set forth in MPEP .sctn.1730
II.B.2(a)(A), and this electronic filing includes an electronically
submitted sequence (SEQ ID) listing. The entire content of this
sequence listing is herein incorporated by reference for all
purposes. The sequence listing is identified on the electronically
filed .txt file as follows:
TABLE-US-00001 File Name Date of Creation Size (bytes)
564462014441seqlist.txt May 23, 2007 367,627 bytes
FIELD OF THE INVENTION
[0002] This invention relates to molecular and cellular biology and
biochemistry. In one aspect, the invention provides polypeptides
having ammonia lyase activity, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase activity,
polynucleotides encoding these polypeptides, and methods of making
and using these polynucleotides and polypeptides. In one aspect,
the invention is directed to polypeptides having ammonia lyase
activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase activity, including thermostable and
thermotolerant activity, and polynucleotides encoding these
enzymes, and making and using these polynucleotides and
polypeptides. The polypeptides of the invention can be used in a
variety of pharmaceutical, agricultural and industrial
contexts.
[0003] Additionally, the polypeptides of the invention can be used
in the synthesis or manufacture of phenylalanine and tyrosine as
well as phenylalanine and tyrosine derivatives. Applications also
include utilizing the enzymes to degrade phenylalanine, tyrosine,
and derivatives thereof to manufacture cinnamic acid,
para-hydroxycinnamic acid, para-hydroxyl styrene and derivatives
thereof. Polypeptides of the invention can also be used in the
synthesis or manufacture of ortho, meta and para isomers of
phenylalanine or related compounds, as well as derivatives thereof.
Polypeptides of the invention can also be used in the synthesis or
manufacture of urocanoic acid and urocanoic acid derivatives, from
histidine and histidine derivatives. Polypeptides of the invention
can also be used in enzyme substitution therapies for the treatment
of phenylketonuria (PKU). Thus, fields of use include manufacture
of bulk and fine chemicals for industrial, medicinal and
agricultural use, as well as the direct application of the enzymes
themselves for enzyme substitution therapy for a variety of
diseases.
BACKGROUND
[0004] Phenylalanine ammonia lyases (PAL, EC 4.3.1.5) catalyze the
deamination of phenylalanine to trans-cinnamic acid and ammonia
(FIG. 5). In nature, they facilitate the committed step in
phenylpropanoid pathways to produce lignins, coumarins, and
flavonoids. Depending on the source of the enzyme, PALs may show
varying selectivity towards phenylalanine and tyrosine derivatives
(those active on tyrosine derivatives are known as tyrosine ammonia
lyases (TALs)). Histidine ammonia lyases (HALs, EC 4.3.1.3) are
distinct from PALs in that they have a substrate preference for
histidine over phenylalanine or tyrosine. HALs catalyze the
abstraction of ammonia from histidine to form urocanoic acid.
[0005] Most of the phenylalanine ammonia lyases (PALs) currently
described are from plant origins where the enzyme plays a central
role in plant metabolism. Recently, PALs have been identified in
fungi and a very limited number have been identified in bacteria.
HALs have also been identified in plants and fungi. Unlike PALs,
HALs have been found to be widespread in bacteria. Synthetic
applications of HALs tend to be rather limited compared to PALs.
Some niche applications have been developed such as the synthesis
of radiolabeled urocanoic acids as tracers of histidine metabolism.
There may be potential to expand applications of HALs by discovery
of enzymes with greater stability to oxygen.
[0006] Up until the late 1990s, it was thought that histidine and
phenylalanine ammonia lyases utilized a dehydroalanine cofactor in
their catalytic mechanism. However X-ray crystallographic studies
have shown that the cofactor is actually
3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO), which is formed
by cyclization and dehydration of a conserved active site
Ala-Ser-Gly sequence. Enzyme mechanistic studies have led to two
main proposals on the catalytic mechanism of phenylalanine ammonia
lyases (PALs), as shown in FIGS. 6a and 6b. In both mechanisms A
and B, the MIO group acts as a powerful electrophile; in mechanism
A the MIO group reacts with the amino group of Phe, while in
mechanism B it reacts with the aromatic side chain in a
Friedel-Crafts-type reaction.
[0007] Applications of PALs include the manufacture of
phenylalanine and tyrosine as well as phenylalanine and tyrosine
derivatives. Applications include utilizing the enzymes to degrade
phenylalanine, tyrosine, and derivatives to manufacture cinnamic
acid, para-hydroxycinnamic acid and derivatives. Fields of use
include manufacture of bulk and fine chemicals for industrial,
medicinal and agricultural use, as well as the direct application
of the enzymes themselves for an enzyme substitution therapy.
[0008] For example, PALs have been investigated for an enzyme
substitution therapy for the treatment of phenylketonuria (PKU), an
inherited metabolic disease caused by a deficiency of the enzyme
phenylalanine hydroxylase. PKU is one of the most commonly
inherited metabolic disorders, affecting an estimated 50,000 people
in the developed world or 30,000 people in the United States. It
occurs in approximately 1 in 10,000 (0.01%) babies born in the US.
PKU is an inborn error of amino acid metabolism caused by a
phenylalanine hydroxylase defect (PAH). Untreated patients with PKU
often show mental retardation or otherwise impaired cognitive
function. Currently the only treatment for PKU is strict dietary
control via a low-phenylalanine diet. A few pharmaceutical
modalities to treat PKU are under investigation. One of these
approaches is the use of phenylalanine ammonia-lyase (PAL) as an
enzyme replacement therapy. Several reports of applying a PAL (R.
toruloides) to decrease phenylalanine serum levels in murine models
have been published. However, developing a form of this enzyme with
sufficiently high activity and stability has proven difficult. One
concept was the application of PAL as an oral treatment to break
down phenylalanine in the gut. PAL therapy is also being considered
for use with CLE.TM. (crystallized enzyme crystal) methodology to
stabilize the enzyme for oral delivery. Degradation of
phenylalanine by PAL treatment yields trans-cinnamate which has
very low toxicity. In addition, PAL therapy has the advantage that
it does not require exogenous cofactors to degrade Phe. There is a
need for more PAL enzymes to extend the utility of this versatile
enzyme class, especially PALs of bacterial origin. Bacterial PALs
potentially offer greater catalytic versatility than plant and
fungal enzymes since their natural cellular roles are likely more
diverse.
SUMMARY
[0009] The invention provides polypeptides, including enzymes,
having ammonia lyase activity, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase activity,
nucleic acids encoding them, antibodies that bind to them, and
methods of making and using them.
[0010] In one aspect, polypeptides of the invention can be used in
the synthesis or manufacture of .alpha.-amino acids and derivatives
or .beta.-amino acids and derivatives, e.g. phenylalanine,
histidine or tyrosine and derivatives thereof. In one aspect, the
.alpha. or .beta.-amino acids synthesized or manufactured using a
polypeptide of the invention include phenylalanine, histidine or
tyrosine and derivatives and analogs thereof, including
phenylalanine, histidine or tyrosine altered by substitution with
(addition of) a halogen-, methyl-, ethyl-, hydroxy-,
hydroxymethyl-, nitro-, or amino-comprising group in any or all of
the 2, 3, 4, and 5 positions in the aromatic side chain of the
amino acid. For example, polypeptides of the invention can be used
in the synthesis or manufacture of ortho, meta and para isomers of
phenylalanine and/or tyrosine, e.g., ortho-, meta- or para-bromo
phenylalanine; ortho-, meta- or para-fluoro phenylalanine; ortho-,
meta- or para-iodo phenylalanine; ortho-, meta- or para-chloro
phenylalanine; ortho-, meta- or para-methyl phenylalanine; ortho-,
meta- or para-hydroxyl phenylalanine; ortho-, meta- or
para-hydroxymethyl phenylalanine; ortho-, meta- or para-ethyl
phenylalanine ortho-, meta- or para-nitro phenylalanine; ortho-,
meta- or para-amino phenylalanine; ortho-, or meta-bromo tyrosine;
ortho- or meta-fluoro tyrosine; ortho- or meta-iodo tyrosine;
ortho-, or meta-chloro tyrosine; ortho- or meta-methyl tyrosine;
ortho- or meta-hydroxyl tyrosine; ortho- or meta-hydroxymethyl
tyrosine; ortho- or meta-ethyl tyrosine; ortho- or meta-nitro
tyrosine; ortho- or meta-amino tyrosine, all in both L and D
enantiomers, such as L- and D-.alpha. or .beta.-amino acids (e.g.,
L-phenylalanine and D-phenylalanine, L- and D-histidine, L- and
D-tyrosine), as well as derivatives thereof. In one aspect, the
invention provides methods for the synthesis or manufacture of L-
and D-phenylalanine and L- and D-tyrosine as well as L- and
D-phenylalanine and L- and D-tyrosine derivatives (see FIG. 5). In
another aspect, the invention provides methods for the synthesis or
manufacture of cinnamic acid and cinnamic acid derivatives. In yet
another aspect, the invention provides methods for the synthesis or
manufacture of para-hydroxycinnamic acid and para-hydroxyl styrene
via biocatalytic and fermentation. In another aspect, the invention
provides methods for the synthesis or manufacture of ortho-bromo
and ortho-chloro L-phenylalanine and of ortho-bromo and
ortho-chloro D-phenylalanine, as well as derivatives thereof. In
yet another aspect, the invention provides methods for the
synthesis or manufacture of L- and D-.beta.-amino acids (see FIG.
7) and L- and D-histidine and derivatives. In another aspect, the
invention provides methods for the synthesis or manufacture of
urocanoic acid and urocanoic acid derivatives, from histidine and
histidine derivatives. In one aspect, the enzymes of the invention
can be used to catalyze the reverse reaction of any of the
reactions described herein.
[0011] In further aspects, the invention provides methods for the
manufacture of bulk and fine chemicals for industrial, medicinal
and agricultural use, using the enzymes of the invention. In other
aspects, the invention provides methods of application of the
enzymes of the invention for enzyme substitution therapy, e.g.,
using PALs for the treatment of phenylketonuria (PKU), an inherited
metabolic disease caused by a deficiency of the enzyme
phenylalanine hydroxylase.
[0012] In one aspect the invention provides compositions (e.g.,
feeds, drugs, dietary supplements) comprising the enzymes,
polypeptides or polynucleotides of the invention.
[0013] These compositions can be formulated in a variety of forms,
e.g., as liquids, sprays, films, micelles, liposomes, powders,
food, feed pellets or encapsulated forms, including encapsulated
forms.
[0014] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary nucleic acid of the
invention, including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ
ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,
SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99 and SEQ ID
NO:101 over a region of at least about 10, 15, 20, 25, 30, 35, 40,
45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,
1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,
1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350,
2400, 2450, 2500, or more residues, wherein the nucleic acid
encodes at least one polypeptide having an ammonia lyase activity,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase activity, or encodes a peptide or
polypeptide that can be used to generate an antibody that
specifically binds to an exemplary polypeptide of the invention
(see below). In one aspect, the sequence identities are determined
by analysis with a sequence comparison algorithm or by a visual
inspection.
[0015] Exemplary nucleic acids of the invention also include
isolated, synthetic or recombinant nucleic acids encoding an
exemplary polypeptide of the invention, including SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ
ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,
SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID
NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ
ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,
SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID
NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ
ID NO:98, SEQ ID NO:100 and SEQ ID NO:102, and subsequences thereof
and variants thereof. In one aspect, the polypeptide has an ammonia
lyase activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase activity, or, the polypeptide
or peptide has immunogenic activity.
[0016] In one aspect, the invention also provides ammonia
lyase-encoding, e.g., phenylalanine ammonia lyase-, tyrosine
ammonia lyase- and/or histidine ammonia lyase-encoding nucleic
acids with a common novelty in that they are derived from mixed
cultures. The invention provides ammonia lyase-degrading
enzyme-encoding nucleic acids isolated from mixed cultures
comprising a polynucleotide of the invention, e.g., a sequence
having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or
complete (100%) sequence identity to an exemplary nucleic acid of
the invention, which includes all the odd numbered sequences from
SEQ ID NO:1 through SEQ ID NO:101, over a region of at least about
50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or more, or,
nucleic acids which encode an enzymatically active fragment of an
exemplary sequence of the invention.
[0017] In one aspect, the invention provides ammonia lyase enzyme-,
e.g., phenylalanine ammonia lyase enzyme-, tyrosine ammonia lyase
enzyme- and/or histidine ammonia lyase enzyme-encoding nucleic
acids with a common novelty in that they are derived from a common
source, e.g., an environmental source. In one aspect, the invention
also provides ammonia lyase enzyme-, e.g., phenylalanine ammonia
lyase enzyme-, tyrosine ammonia lyase enzyme- and/or histidine
ammonia lyase enzyme-encoding nucleic acids with a common novelty
in that they are derived from environmental sources, e.g., mixed
environmental sources.
[0018] In one aspect, the sequence comparison algorithm is a BLAST
version 2.2.2 algorithm where a filtering setting is set to
blastall -p blastp -d "nr pataa"-F F, and all other options are set
to default.
[0019] Another aspect of the invention is an isolated or
recombinant nucleic acid including at least 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,
1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300,
2350, 2400, 2450, 2500, or more consecutive bases of a nucleic acid
sequence of the invention, sequences substantially identical
thereto, and the sequences complementary thereto.
[0020] In one aspect, the isolated or recombinant nucleic acid
encodes a polypeptide having an ammonia lyase activity, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase activity, which is thermostable. The
polypeptide can retain an ammonia lyase activity under conditions
comprising a temperature range of between about 37.degree. C. to
about 95.degree. C.; between about 55.degree. C. to about
85.degree. C., between about 70.degree. C. to about 95.degree. C.,
or, between about 90.degree. C. to about 95.degree. C.
[0021] In another aspect, the isolated or recombinant nucleic acid
encodes a polypeptide having an ammonia lyase activity, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase activity, which is thermotolerant. The
polypeptide can retain an ammonia lyase activity after exposure to
a temperature in the range from greater than 37.degree. C. to about
95.degree. C. or anywhere in the range from greater than 55.degree.
C. to about 85.degree. C. The polypeptide can retain an ammonia
lyase activity after exposure to a temperature in the range between
about 1.degree. C. to about 5.degree. C., between about 5.degree.
C. to about 15.degree. C., between about 15.degree. C. to about
25.degree. C., between about 25.degree. C. to about 37.degree. C.,
between about 37.degree. C. to about 95.degree. C., between about
55.degree. C. to about 85.degree. C., between about 70.degree. C.
to about 75.degree. C., or between about 90.degree. C. to about
95.degree. C., or more. In one aspect, the polypeptide retains an
ammonia lyase activity after exposure to a temperature in the range
from greater than 90.degree. C. to about 95.degree. C. at about pH
4.5.
[0022] The invention provides isolated, synthetic or recombinant
nucleic acids comprising a sequence that hybridizes under stringent
conditions to a nucleic acid comprising a sequence of the
invention, e.g., an exemplary sequence of the invention, e.g., as
set forth in SEQ ID NO:1 through SEQ ID NO:101, or fragments or
subsequences thereof. In one aspect, the nucleic acid encodes a
polypeptide having an ammonia lyase activity, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase activity. The nucleic acid can be at least about 10, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1150, 1200 or more residues in length or the full length of the
gene or transcript. In one aspect, the stringent conditions include
a wash step comprising a wash in 0.2.times.SSC at a temperature of
about 65.degree. C. for about 15 minutes.
[0023] The invention provides a nucleic acid probe for identifying
a nucleic acid encoding a polypeptide having an ammonia lyase
activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase activity, wherein the probe
comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or
more, consecutive bases of a sequence comprising a sequence of the
invention, or fragments or subsequences thereof, wherein the probe
identifies the nucleic acid by binding or hybridization. The probe
can comprise an oligonucleotide comprising at least about 10 to 50,
about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100
consecutive bases of a sequence comprising a sequence of the
invention, or fragments or subsequences thereof.
[0024] The invention provides a nucleic acid probe for identifying
a nucleic acid encoding a polypeptide having an ammonia lyase
activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase activity, wherein the probe
comprises a nucleic acid comprising a sequence at least about 10,
15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or
more residues having at least about 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to a nucleic acid of the invention. In one aspect, the
sequence identities are determined by analysis with a sequence
comparison algorithm or by visual inspection. In alternative
aspects, the probe can comprise an oligonucleotide comprising at
least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to
80, or about 60 to 100, or about 50 to 150, or about 100 to 200,
consecutive bases of a nucleic acid sequence of the invention, or a
subsequence thereof.
[0025] The invention provides an amplification primer pair for
amplifying a nucleic acid encoding a polypeptide having an ammonia
lyase activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase activity, wherein the primer
pair is capable of amplifying a nucleic acid comprising a sequence
of the invention, or fragments or subsequences thereof. One or each
member of the amplification primer sequence pair can comprise an
oligonucleotide comprising at least about 10 to 50, or more,
consecutive bases of the sequence, or about 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
consecutive bases of the sequence.
[0026] The invention provides amplification primer pairs, wherein
the primer pair comprises a first member having a sequence as set
forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36
or more residues of a nucleic acid of the invention, and a second
member having a sequence as set forth by about the first (the 5')
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36 or more residues of the
complementary strand of the first member.
[0027] The invention provides ammonia lyase-encoding, e.g.,
phenylalanine ammonia lyase-, tyrosine ammonia lyase- and/or
histidine ammonia lyase-encoding nucleic acids generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
ammonia lyase-encoding, e.g., phenylalanine ammonia lyase-,
tyrosine ammonia lyase- and/or histidine ammonia lyase-encoding
nucleic acids generated by amplification, e.g., polymerase chain
reaction (PCR), using an amplification primer pair of the
invention. The invention provides methods of making an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme by amplification, e.g.,
polymerase chain reaction (PCR), using an amplification primer pair
of the invention. In one aspect, the amplification primer pair
amplifies a nucleic acid from a library, e.g., a gene library, such
as an environmental library.
[0028] The invention provides methods of amplifying a nucleic acid
encoding a polypeptide having an ammonia lyase activity, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase activity comprising amplification of a
template nucleic acid with an amplification primer sequence pair
capable of amplifying a nucleic acid sequence of the invention, or
fragments or subsequences thereof.
[0029] The invention provides expression cassettes comprising a
nucleic acid of the invention or a subsequence thereof. In one
aspect, the expression cassette can comprise the nucleic acid that
is operably linked to a promoter. The promoter can be a viral,
fungal, yeast, bacterial, mammalian or plant promoter. In one
aspect, the plant promoter can be a potato, rice, corn, wheat,
tobacco or barley promoter. The promoter can be a constitutive
promoter. The constitutive promoter can comprise CaMV35S. In
another aspect, the promoter can be an inducible promoter. In one
aspect, the promoter can be a tissue-specific promoter or an
environmentally regulated or a developmentally regulated promoter.
Thus, the promoter can be, e.g., a seed-specific, a leaf-specific,
a root-specific, a stem-specific or an abscission-induced promoter.
In one aspect, the expression cassette can further comprise a plant
or plant virus expression vector.
[0030] The invention provides cloning vehicles comprising an
expression cassette (e.g., a vector) of the invention or a nucleic
acid of the invention. The cloning vehicle can be a viral vector, a
plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage
or an artificial chromosome. The viral vector can comprise an
adenovirus vector, a retroviral vector or an adeno-associated viral
vector. The cloning vehicle can comprise a bacterial artificial
chromosome (BAC), a plasmid, a bacteriophage P1-derived vector
(PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
[0031] The invention provides transformed cell comprising a nucleic
acid of the invention or an expression cassette (e.g., a vector) of
the invention, or a cloning vehicle of the invention. In one
aspect, the transformed cell can be a bacterial cell, a mammalian
cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
In one aspect, the plant cell can be a cereal, a potato, wheat,
rice, corn, tobacco or barley cell.
[0032] The invention provides transgenic non-human animals
comprising a nucleic acid of the invention or an expression
cassette (e.g., a vector) of the invention. In one aspect, the
animal is a mouse, a rat, a pig, a goat or a sheep.
[0033] The invention provides transgenic plants comprising a
nucleic acid of the invention or an expression cassette (e.g., a
vector) of the invention. The transgenic plant can be a cereal
plant, a corn plant, a potato plant, a tomato plant, a wheat plant,
an oilseed plant, a rapeseed plant, a soybean plant, a rice plant,
a barley plant or a tobacco plant.
[0034] The invention provides transgenic seeds comprising a nucleic
acid of the invention or an expression cassette (e.g., a vector) of
the invention. The transgenic seed can be a cereal plant, a corn
seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a
palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco
plant seed.
[0035] The invention provides an antisense oligonucleotide
comprising a nucleic acid sequence complementary to or capable of
hybridizing under stringent conditions to a nucleic acid of the
invention. The invention provides methods of inhibiting the
translation of an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
message in a cell comprising administering to the cell or
expressing in the cell an antisense oligonucleotide comprising a
nucleic acid sequence complementary to or capable of hybridizing
under stringent conditions to a nucleic acid of the invention. In
one aspect, the antisense oligonucleotide is between about 10 to
50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to
100 bases in length, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases in length.
[0036] The invention provides methods of inhibiting the translation
of an ammonia lyase enzyme, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
message in a cell comprising administering to the cell or
expressing in the cell an antisense oligonucleotide comprising a
nucleic acid sequence complementary to or capable of hybridizing
under stringent conditions to a nucleic acid of the invention. The
invention provides double-stranded inhibitory RNA (RNAi, or RNA
interference) molecules (including small interfering RNA, or
siRNAs, for inhibiting transcription, and microRNAs, or miRNAs, for
inhibiting translation) comprising a subsequence of a sequence of
the invention. In one aspect, the siRNA is between about 21 to 24
residues, or, about at least 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100 or more duplex nucleotides in
length. The invention provides methods of inhibiting the expression
of an ammonia lyase enzyme, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme in a
cell comprising administering to the cell or expressing in the cell
a double-stranded inhibitory RNA (siRNA or miRNA), wherein the RNA
comprises a subsequence of a sequence of the invention.
[0037] The invention provides an isolated or recombinant
polypeptide comprising an amino acid sequence having at least about
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or
complete (100%) sequence identity to an exemplary polypeptide or
peptide of the invention over a region of at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 or more
residues, or over the full length of the polypeptide. In one
aspect, the sequence identities are determined by analysis with a
sequence comparison algorithm or by a visual inspection. Exemplary
polypeptide or peptide sequences of the invention include SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ
ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86,
SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100 and SEQ ID NO:102, and
subsequences thereof and variants thereof. Exemplary polypeptides
also include fragments of at least about 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300,
350, 400, 450, 500, 550, 600 or more residues in length, or over
the full length of an enzyme. Exemplary polypeptide or peptide
sequences of the invention include sequence encoded by a nucleic
acid of the invention. Exemplary polypeptide or peptide sequences
of the invention include polypeptides or peptides specifically
bound by an antibody of the invention, or a peptide or polypeptide
has immunogenic activity, e.g., the peptide or polypeptide can be
used to generate an antibody that specifically binds to an
exemplary polypeptide of the invention.
[0038] In one aspect, a polypeptide of the invention has at least
one ammonia lyase enzyme, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity. In alternative aspects, a polynucleotide of the invention
encodes a polypeptide that has at least one ammonia lyase enzyme,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity.
[0039] In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity is thermostable. The polypeptide can retain
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity under
conditions comprising a temperature range of between about
1.degree. C. to about 5.degree. C., between about 5.degree. C. to
about 15.degree. C., between about 15.degree. C. to about
25.degree. C., between about 25.degree. C. to about 37.degree. C.,
between about 37.degree. C. to about 95.degree. C., between about
55.degree. C. to about 85.degree. C., between about 70.degree. C.
to about 75.degree. C., or between about 90.degree. C. to about
95.degree. C., or more.
[0040] In another aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity can be thermotolerant. The polypeptide can
retain an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity after exposure to a temperature in the range from greater
than 37.degree. C. to about 95.degree. C., or in the range from
greater than 55.degree. C. to about 85.degree. C. The polypeptide
can retain an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity after exposure to conditions comprising a temperature
range of between about 1.degree. C. to about 5.degree. C., between
about 5.degree. C. to about 15.degree. C., between about 15.degree.
C. to about 25.degree. C., between about 25.degree. C. to about
37.degree. C., between about 37.degree. C. to about 95.degree. C.,
between about 55.degree. C. to about 85.degree. C., between about
70.degree. C. to about 75.degree. C., or between about 90.degree.
C. to about 95.degree. C., or more. In one aspect, the polypeptide
can retain an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity after exposure to a temperature in the range from greater
than 90.degree. C. to about 95.degree. C. at pH 4.5.
[0041] Another aspect of the invention provides an isolated or
recombinant polypeptide or peptide including at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100
or more consecutive bases of a polypeptide or peptide sequence of
the invention, sequences substantially identical thereto, and the
sequences complementary thereto. The peptide can be, e.g., an
immunogenic fragment, a motif (e.g., a binding site), a signal
sequence, a prepro sequence or an active site.
[0042] The invention provides isolated or recombinant nucleic acids
comprising a sequence encoding a polypeptide having an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme activity and a signal
sequence, wherein the nucleic acid comprises a sequence of the
invention. By a "signal sequence" is meant a secretion signal or
other domain that facilitates secretion of a polypeptide, e.g., a
lyase, of the invention from the host cell. The signal sequence can
be derived from another enzyme (e.g., another ammonia lyase,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme; or the signal sequence can be
derived from a non-ammonia lyase, e.g., non-phenylalanine ammonia
lyase, non-tyrosine ammonia lyase and/or non-histidine ammonia
lyase enzyme; or, a completely heterologous enzyme. The invention
provides isolated or recombinant nucleic acids comprising a
sequence encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity, wherein the sequence does
not contain a signal sequence and the nucleic acid comprises a
sequence of the invention. In one aspect, the invention provides an
isolated or recombinant polypeptide comprising a polypeptide of the
invention lacking all or part of a signal sequence. In one aspect,
the isolated or recombinant polypeptide can comprise the
polypeptide of the invention comprising a heterologous signal
sequence, such as a heterologous ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme signal sequence or non-ammonia lyase, e.g.,
non-phenylalanine ammonia lyase, non-tyrosine ammonia lyase and/or
non-histidine ammonia lyase enzyme signal sequence.
[0043] In one aspect, the invention provides chimeric proteins
comprising a first domain comprising a signal sequence of the
invention and at least a second domain. The protein can be a fusion
protein. The second domain can comprise an enzyme (where in one
aspect the first domain is a polypeptide of the invention). The
protein can be a non-enzyme.
[0044] The invention provides chimeric polypeptides comprising at
least a first domain comprising signal peptide (SP), a prepro
sequence and/or a catalytic domain (CD) of the invention and at
least a second domain comprising a heterologous polypeptide or
peptide, wherein the heterologous polypeptide or peptide is not
naturally associated with the signal peptide (SP), prepro sequence
and/or catalytic domain (CD). In one aspect, the heterologous
polypeptide or peptide is not an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme. The heterologous polypeptide or peptide can be amino
terminal to, carboxy terminal to or on both ends of the signal
peptide (SP), prepro sequence and/or catalytic domain (CD).
[0045] The invention provides isolated or recombinant nucleic acids
encoding a chimeric polypeptide, wherein the chimeric polypeptide
comprises at least a first domain comprising signal peptide (SP), a
prepro domain and/or a catalytic domain (CD) of the invention and
at least a second domain comprising a heterologous polypeptide or
peptide, wherein the heterologous polypeptide or peptide is not
naturally associated with the signal peptide (SP), prepro domain
and/or catalytic domain (CD).
[0046] The invention provides isolated or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a
sequence as set forth in residues 1 to 10, 1 to 11, 1 to 12, 1 to
13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20,
1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to
28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35,
1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to
44, 1 to 45, 1 to 46 or 1 to 47, of a polypeptide of the invention,
e.g., an exemplary polypeptide of the invention, including all even
numbered sequences between SEQ ID NO:2 and SEQ ID NO:102. In one
aspect, the invention provides signal sequences comprising the
first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70 or more amino terminal residues of a
polypeptide of the invention.
[0047] In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity comprises a specific activity at about
37.degree. C. in the range from about 1 to about 1200 units per
milligram of protein, or, about 100 to about 1000 units per
milligram of protein. In another aspect, the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity comprises a specific
activity from about 100 to about 1000 units per milligram of
protein, or, from about 500 to about 750 units per milligram of
protein. Alternatively, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity comprises a specific activity at 37.degree.
C. in the range from about 1 to about 750 units per milligram of
protein, or, from about 500 to about 1200 units per milligram of
protein. In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity comprises a specific activity at 37.degree.
C. in the range from about 1 to about 500 units per milligram of
protein, or, from about 750 to about 1000 units per milligram of
protein. In another aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity comprises a specific activity at 37.degree.
C. in the range from about 1 to about 250 units per milligram of
protein. Alternatively, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity comprises a specific activity at 37.degree.
C. in the range from about 1 to about 100 units per milligram of
protein.
[0048] In another aspect, the thermotolerance comprises retention
of at least half of the specific activity of the ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme at 37.degree. C. after being heated
to the elevated temperature. Alternatively, the thermotolerance can
comprise retention of specific activity at 37.degree. C. in the
range from about 1 to about 1200 units per milligram of protein,
or, from about 500 to about 1000 units per milligram of protein,
after being heated to the elevated temperature. In another aspect,
the thermotolerance can comprise retention of specific activity at
37.degree. C. in the range from about 1 to about 500 units per
milligram of protein after being heated to the elevated
temperature.
[0049] The invention provides the isolated or recombinant
polypeptide of the invention, wherein the polypeptide comprises at
least one glycosylation site. In one aspect, glycosylation can be
an N-linked glycosylation. In one aspect, the polypeptide can be
glycosylated after being expressed in a P. pastoris or a S. pombe
host or in any mammalian, fungal, bacterial, insect, yeast or other
host cell.
[0050] In one aspect, the polypeptide can retain ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity under conditions comprising
about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another
aspect, the polypeptide can retain an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity under conditions comprising
about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or
pH 11. In one aspect, the polypeptide can retain an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity after exposure to
conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or
pH 4 or more acidic conditions. In another aspect, the polypeptide
can retain an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity after exposure to conditions comprising about pH 7, pH 7.5
pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more
alkaline conditions.
[0051] In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme of the invention has activity at under alkaline
conditions, e.g., the alkaline conditions of the gut, e.g., the
small intestine. In one aspect, the polypeptide can retains
activity after exposure to the acidic pH of the stomach.
[0052] The invention provides protein preparations comprising a
polypeptide of the invention, wherein the protein preparation
comprises a liquid, a solid or a gel.
[0053] The invention provides heterodimers comprising a polypeptide
of the invention and a second protein or domain. The second member
of the heterodimer can be a different ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme, a different enzyme or another
protein. In one aspect, the second domain can be a polypeptide and
the heterodimer can be a fusion protein. In one aspect, the second
domain can be an epitope or a tag. In one aspect, the invention
provides homomultimers, including, but not limited to, homodimers,
homotrimers, homotetramers, homopentamers, and homohexamers, etc.,
comprising a polypeptide of the invention.
[0054] The invention provides immobilized polypeptides having
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme activity, wherein the
polypeptide comprises a polypeptide of the invention, a polypeptide
encoded by a nucleic acid of the invention, or a polypeptide
comprising a polypeptide of the invention and a second domain. In
one aspect, the polypeptide can be immobilized on a cell, a metal,
a resin, a polymer, a ceramic, a glass, a microelectrode, a
graphitic particle, a bead, a gel, a plate, an array or a capillary
tube.
[0055] The invention provides arrays comprising an immobilized
nucleic acid of the invention. The invention provides arrays
comprising an antibody of the invention.
[0056] The invention provides isolated or recombinant antibodies
that specifically bind to a polypeptide of the invention or to a
polypeptide encoded by a nucleic acid of the invention. These
antibodies of the invention can be a monoclonal or a polyclonal
antibody. The invention provides hybridomas comprising an antibody
of the invention, e.g., an antibody that specifically binds to a
polypeptide of the invention or to a polypeptide encoded by a
nucleic acid of the invention. The invention provides nucleic acids
encoding these antibodies.
[0057] The invention provides method of isolating or identifying a
polypeptide having ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity comprising the steps of: (a) providing an antibody of the
invention; (b) providing a sample comprising polypeptides; and (c)
contacting the sample of step (b) with the antibody of step (a)
under conditions wherein the antibody can specifically bind to the
polypeptide, thereby isolating or identifying a polypeptide having
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity.
[0058] The invention provides methods of making an anti-ammonia
lyase, e.g., anti-phenylalanine ammonia lyase, anti-tyrosine
ammonia lyase and/or anti-histidine ammonia lyase enzyme antibody
comprising administering to a non-human animal a nucleic acid of
the invention or a polypeptide of the invention or subsequences
thereof in an amount sufficient to generate a humoral immune
response, thereby making an anti-ammonia lyase, e.g.,
anti-phenylalanine ammonia lyase, anti-tyrosine ammonia lyase
and/or anti-histidine ammonia lyase enzyme antibody. The invention
provides methods of making an anti-ammonia lyase, e.g.,
anti-phenylalanine ammonia lyase, anti-tyrosine ammonia lyase
and/or anti-histidine ammonia lyase enzyme immune comprising
administering to a non-human animal a nucleic acid of the invention
or a polypeptide of the invention or subsequences thereof in an
amount sufficient to generate an immune response.
[0059] The invention provides methods of producing a recombinant
polypeptide comprising the steps of: (a) providing a nucleic acid
of the invention operably linked to a promoter; and (b) expressing
the nucleic acid of step (a) under conditions that allow expression
of the polypeptide, thereby producing a recombinant polypeptide. In
one aspect, the method can further comprise transforming a host
cell with the nucleic acid of step (a) followed by expressing the
nucleic acid of step (a), thereby producing a recombinant
polypeptide in a transformed cell.
[0060] The invention provides methods for identifying a polypeptide
having ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity
comprising the following steps: (a) providing a polypeptide of the
invention; or a polypeptide encoded by a nucleic acid of the
invention; (b) providing ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
substrate; and (c) contacting the polypeptide or a fragment or
variant thereof of step (a) with the substrate of step (b) and
detecting a decrease in the amount of substrate or an increase in
the amount of a reaction product, wherein a decrease in the amount
of the substrate or an increase in the amount of the reaction
product detects a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity. In one aspect, the
substrate is a histidine-, phenylalanine- or tyrosine-comprising
compound.
[0061] The invention provides methods for identifying ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme substrate comprising the
following steps: (a) providing a polypeptide of the invention; or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing a test substrate; and (c) contacting the polypeptide of
step (a) with the test substrate of step (b) and detecting a
decrease in the amount of substrate or an increase in the amount of
reaction product, wherein a decrease in the amount of the substrate
or an increase in the amount of a reaction product identifies the
test substrate as an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
substrate.
[0062] The invention provides methods of determining whether a test
compound specifically binds to a polypeptide comprising the
following steps: (a) expressing a nucleic acid or a vector
comprising the nucleic acid under conditions permissive for
translation of the nucleic acid to a polypeptide, wherein the
nucleic acid comprises a nucleic acid of the invention, or,
providing a polypeptide of the invention; (b) providing a test
compound; (c) contacting the polypeptide with the test compound;
and (d) determining whether the test compound of step (b)
specifically binds to the polypeptide.
[0063] The invention provides methods for identifying a modulator
of an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity
comprising the following steps: (a) providing a polypeptide of the
invention or a polypeptide encoded by a nucleic acid of the
invention; (b) providing a test compound; (c) contacting the
polypeptide of step (a) with the test compound of step (b) and
measuring an activity of the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme, wherein a change in the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity measured in the presence of
the test compound compared to the activity in the absence of the
test compound provides a determination that the test compound
modulates the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity. In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity can be measured by providing an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme substrate and detecting a
decrease in the amount of the substrate or an increase in the
amount of a reaction product, or, an increase in the amount of the
substrate or a decrease in the amount of a reaction product. A
decrease in the amount of the substrate or an increase in the
amount of the reaction product with the test compound as compared
to the amount of substrate or reaction product without the test
compound identifies the test compound as an activator of ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme activity. An increase in the
amount of the substrate or a decrease in the amount of the reaction
product with the test compound as compared to the amount of
substrate or reaction product without the test compound identifies
the test compound as an inhibitor of ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity.
[0064] The invention provides computer systems comprising a
processor and a data storage device wherein said data storage
device has stored thereon a polypeptide sequence or a nucleic acid
sequence of the invention (e.g., a polypeptide encoded by a nucleic
acid of the invention). In one aspect, the computer system can
further comprise a sequence comparison algorithm and a data storage
device having at least one reference sequence stored thereon. In
another aspect, the sequence comparison algorithm comprises a
computer program that indicates polymorphisms. In one aspect, the
computer system can further comprise an identifier that identifies
one or more features in said sequence. The invention provides
computer readable media having stored thereon a polypeptide
sequence or a nucleic acid sequence of the invention. The invention
provides methods for identifying a feature in a sequence comprising
the steps of: (a) reading the sequence using a computer program
which identifies one or more features in a sequence, wherein the
sequence comprises a polypeptide sequence or a nucleic acid
sequence of the invention; and (b) identifying one or more features
in the sequence with the computer program. The invention provides
methods for comparing a first sequence to a second sequence
comprising the steps of: (a) reading the first sequence and the
second sequence through use of a computer program which compares
sequences, wherein the first sequence comprises a polypeptide
sequence or a nucleic acid sequence of the invention; and (b)
determining differences between the first sequence and the second
sequence with the computer program. The step of determining
differences between the first sequence and the second sequence can
further comprise the step of identifying polymorphisms. In one
aspect, the method can further comprise an identifier that
identifies one or more features in a sequence. In another aspect,
the method can comprise reading the first sequence using a computer
program and identifying one or more features in the sequence.
[0065] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity from a sample, such as an
environmental sample comprising the steps of: (a) providing an
amplification primer sequence pair for amplifying a nucleic acid
encoding a polypeptide having an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity, wherein the primer pair is capable of
amplifying a nucleic acid of the invention; (b) isolating a nucleic
acid from the sample or treating the sample such that nucleic acid
in the sample is accessible for hybridization to the amplification
primer pair; and, (c) combining the nucleic acid of step (b) with
the amplification primer pair of step (a) and amplifying nucleic
acid from the sample, thereby isolating or recovering a nucleic
acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity from a sample. One or each
member of the amplification primer sequence pair can comprise an
oligonucleotide comprising an amplification primer sequence pair of
the invention, e.g., having at least about 10 to 50 consecutive
bases of a sequence of the invention. In one embodiment of the
invention, the sample is an environmental sample.
[0066] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity from a sample, such as an
environmental sample, comprising the steps of: (a) providing a
polynucleotide probe comprising a nucleic acid of the invention or
a subsequence thereof; (b) isolating a nucleic acid from the sample
or treating the sample such that nucleic acid in the sample is
accessible for hybridization to a polynucleotide probe of step (a);
(c) combining the isolated nucleic acid or the treated sample of
step (b) with the polynucleotide probe of step (a); and (d)
isolating a nucleic acid that specifically hybridizes with the
polynucleotide probe of step (a), thereby isolating or recovering a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity from the sample. The sample
can comprise an environmental sample, e.g., a water sample, a
liquid sample, a soil sample, an air sample or a biological sample.
In one aspect, the biological sample can be derived from a
bacterial cell, a protozoan cell, an insect cell, a yeast cell, a
plant cell, a fungal cell or a mammalian cell. In one embodiment of
the invention, the sample is an environmental sample.
[0067] The invention provides methods of generating a variant of a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity comprising the steps of:
(a) providing a template nucleic acid comprising a nucleic acid of
the invention; and (b) modifying, deleting or adding one or more
nucleotides in the template sequence, or a combination thereof, to
generate a variant of the template nucleic acid. In one aspect, the
method can further comprise expressing the variant nucleic acid to
generate a variant ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
polypeptide. The modifications, additions or deletions can be
introduced by a method comprising error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a
combination thereof. In another aspect, the modifications,
additions or deletions are introduced by a method comprising
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0068] In one aspect, the method can be iteratively repeated until
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme having an
altered or different activity or an altered or different stability
from that of a polypeptide encoded by the template nucleic acid is
produced. In one aspect, the variant ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme polypeptide is thermotolerant, and
retains some activity after being exposed to an elevated
temperature. In another aspect, the variant ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme polypeptide has increased
glycosylation as compared to the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme encoded by a template nucleic acid. Alternatively, the
variant ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme polypeptide has
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity under
a high temperature, wherein the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme encoded by the template nucleic acid is not active
under the high temperature. In one aspect, the method can be
iteratively repeated until an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme coding sequence having an altered codon usage from
that of the template nucleic acid is produced. In another aspect,
the method can be iteratively repeated until an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme gene having higher or lower level of
message expression or stability from that of the template nucleic
acid is produced.
[0069] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity to increase its expression
in a host cell, the method comprising the following steps: (a)
providing a nucleic acid of the invention encoding a polypeptide
having an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity; and, (b) identifying a non-preferred or a less preferred
codon in the nucleic acid of step (a) and replacing it with a
preferred or neutrally used codon encoding the same amino acid as
the replaced codon, wherein a preferred codon is a codon
over-represented in coding sequences in genes in the host cell and
a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to increase its expression in a
host cell.
[0070] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity; the method comprising the
following steps: (a) providing a nucleic acid of the invention;
and, (b) identifying a codon in the nucleic acid of step (a) and
replacing it with a different codon encoding the same amino acid as
the replaced codon, thereby modifying codons in a nucleic acid
encoding an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme.
[0071] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity to increase its expression
in a host cell, the method comprising the following steps: (a)
providing a nucleic acid of the invention encoding an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme polypeptide; and, (b)
identifying a non-preferred or a less preferred codon in the
nucleic acid of step (a) and replacing it with a preferred or
neutrally used codon encoding the same amino acid as the replaced
codon, wherein a preferred codon is a codon over-represented in
coding sequences in genes in the host cell and a non-preferred or
less preferred codon is a codon under-represented in coding
sequences in genes in the host cell, thereby modifying the nucleic
acid to increase its expression in a host cell.
[0072] The invention provides methods for modifying a codon in a
nucleic acid encoding a polypeptide having an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity to decrease its expression
in a host cell, the method comprising the following steps: (a)
providing a nucleic acid of the invention; and (b) identifying at
least one preferred codon in the nucleic acid of step (a) and
replacing it with a non-preferred or less preferred codon encoding
the same amino acid as the replaced codon, wherein a preferred
codon is a codon over-represented in coding sequences in genes in a
host cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to decrease its expression in a
host cell. In one aspect, the host cell can be a bacterial cell, a
fungal cell, an insect cell, a yeast cell, a plant cell or a
mammalian cell.
[0073] The invention provides methods for producing a library of
nucleic acids encoding a plurality of modified ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme active sites or substrate binding
sites, wherein the modified active sites or substrate binding sites
are derived from a first nucleic acid comprising a sequence
encoding a first active site or a first substrate binding site the
method comprising the following steps: (a) providing a first
nucleic acid encoding a first active site or first substrate
binding site, wherein the first nucleic acid sequence comprises a
sequence that hybridizes under stringent conditions to a nucleic
acid of the invention, and the nucleic acid encodes an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme active site or an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme substrate binding site; (b)
providing a set of mutagenic oligonucleotides that encode
naturally-occurring amino acid variants at a plurality of targeted
codons in the first nucleic acid; and, (c) using the set of
mutagenic oligonucleotides to generate a set of active
site-encoding or substrate binding site-encoding variant nucleic
acids encoding a range of amino acid variations at each amino acid
codon that was mutagenized, thereby producing a library of nucleic
acids encoding a plurality of modified ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme active sites or substrate binding
sites. In one aspect, the method comprises mutagenizing the first
nucleic acid of step (a) by a method comprising an optimized
directed evolution system, Gene Site Saturation Mutagenesis (GSSM),
synthetic ligation reassembly (SLR), error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, and a combination
thereof. In another aspect, the method comprises mutagenizing the
first nucleic acid of step (a) or variants by a method comprising
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0074] The invention provides methods for making a small molecule
comprising the following steps: (a) providing a plurality of
biosynthetic enzymes capable of synthesizing or modifying a small
molecule, wherein one of the enzymes comprises an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme encoded by a nucleic acid of the
invention; (b) providing a substrate for at least one of the
enzymes of step (a); and (c) reacting the substrate of step (b)
with the enzymes under conditions that facilitate a plurality of
biocatalytic reactions to generate a small molecule by a series of
biocatalytic reactions. The invention provides methods for
modifying a small molecule comprising the following steps: (a)
providing an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme,
wherein the enzyme comprises a polypeptide of the invention, or, a
polypeptide encoded by a nucleic acid of the invention, or a
subsequence thereof; (b) providing a small molecule; and (c)
reacting the enzyme of step (a) with the small molecule of step (b)
under conditions that facilitate an enzymatic reaction catalyzed by
the ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme, thereby
modifying a small molecule by an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymatic reaction. In one aspect, the method can comprise a
plurality of small molecule substrates for the enzyme of step (a),
thereby generating a library of modified small molecules produced
by at least one enzymatic reaction catalyzed by the ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme. In one aspect, the method can
comprise a plurality of additional enzymes under conditions that
facilitate a plurality of biocatalytic reactions by the enzymes to
form a library of modified small molecules produced by the
plurality of enzymatic reactions. In another aspect, the method can
further comprise the step of testing the library to determine if a
particular modified small molecule that exhibits a desired activity
is present within the library. The step of testing the library can
further comprise the steps of systematically eliminating all but
one of the biocatalytic reactions used to produce a portion of the
plurality of the modified small molecules within the library by
testing the portion of the modified small molecule for the presence
or absence of the particular modified small molecule with a desired
activity, and identifying at least one specific biocatalytic
reaction that produces the particular modified small molecule of
desired activity.
[0075] The invention provides methods for determining a functional
fragment of an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
comprising the steps of: (a) providing an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme, wherein the enzyme comprises a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention, or a subsequence thereof; and (b) deleting a
plurality of amino acid residues from the sequence of step (a) and
testing the remaining subsequence for an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity, thereby determining a
functional fragment of an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme. In one aspect, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity is measured by providing an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme substrate and detecting a decrease
in the amount of the substrate or an increase in the amount of a
reaction product.
[0076] The invention provides methods for whole cell engineering of
new or modified phenotypes by using real-time metabolic flux
analysis, the method comprising the following steps: (a) making a
modified cell by modifying the genetic composition of a cell,
wherein the genetic composition is modified by addition to the cell
of a nucleic acid of the invention; (b) culturing the modified cell
to generate a plurality of modified cells; (c) measuring at least
one metabolic parameter of the cell by monitoring the cell culture
of step (b) in real time; and, (d) analyzing the data of step (c)
to determine if the measured parameter differs from a comparable
measurement in an unmodified cell under similar conditions, thereby
identifying an engineered phenotype in the cell using real-time
metabolic flux analysis. In one aspect, the genetic composition of
the cell can be modified by a method comprising deletion of a
sequence or modification of a sequence in the cell, or, knocking
out the expression of a gene. In one aspect, the method can further
comprise selecting a cell comprising a newly engineered phenotype.
In another aspect, the method can comprise culturing the selected
cell, thereby generating a new cell strain comprising a newly
engineered phenotype.
[0077] The invention provides methods of increasing thermotolerance
or thermostability of an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
polypeptide, the method comprising glycosylating an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme polypeptide, wherein the polypeptide
comprises at least thirty contiguous amino acids of a polypeptide
of the invention; or a polypeptide encoded by a nucleic acid
sequence of the invention, thereby increasing the thermotolerance
or thermostability of the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase polypeptide. In one aspect, the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme specific activity can be
thermostable or thermotolerant at a temperature in the range from
greater than about 37.degree. C. to about 95.degree. C.
[0078] The invention provides methods for overexpressing a
recombinant ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase polypeptide
in a cell comprising expressing a vector comprising a nucleic acid
comprising a nucleic acid of the invention or a nucleic acid
sequence of the invention, wherein the sequence identities are
determined by analysis with a sequence comparison algorithm or by
visual inspection, wherein overexpression is effected by use of a
high activity promoter, a dicistronic vector or by gene
amplification of the vector.
[0079] The invention provides methods of making a transgenic plant
comprising the following steps: (a) introducing a heterologous
nucleic acid sequence into the cell, wherein the heterologous
nucleic sequence comprises a nucleic acid sequence of the
invention, thereby producing a transformed plant cell; and (b)
producing a transgenic plant from the transformed cell. In one
aspect, the step (a) can further comprise introducing the
heterologous nucleic acid sequence by electroporation or
microinjection of plant cell protoplasts. In another aspect, the
step (a) can further comprise introducing the heterologous nucleic
acid sequence directly to plant tissue by DNA particle bombardment.
Alternatively, the step (a) can further comprise introducing the
heterologous nucleic acid sequence into the plant cell DNA using an
Agrobacterium tumefaciens host. In one aspect, the plant cell can
be a potato, corn, rice, wheat, tobacco, or barley cell.
[0080] The invention provides methods of expressing a heterologous
nucleic acid sequence in a plant cell comprising the following
steps: (a) transforming the plant cell with a heterologous nucleic
acid sequence operably linked to a promoter, wherein the
heterologous nucleic sequence comprises a nucleic acid of the
invention; (b) growing the plant under conditions wherein the
heterologous nucleic acids sequence is expressed in the plant cell.
The invention provides methods of expressing a heterologous nucleic
acid sequence in a plant cell comprising the following steps: (a)
transforming the plant cell with a heterologous nucleic acid
sequence operably linked to a promoter, wherein the heterologous
nucleic sequence comprises a sequence of the invention; (b) growing
the plant under conditions wherein the heterologous nucleic acids
sequence is expressed in the plant cell.
[0081] The invention provides feeds or foods comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention. In one aspect, the invention provides a
food, feed, a liquid, e.g., a beverage (such as a fruit juice or a
beer), a bread or a dough or a bread product, or a beverage
precursor (e.g., a wort), comprising a polypeptide of the
invention. The invention provides food or nutritional supplements
for an animal comprising a polypeptide of the invention, e.g., a
polypeptide encoded by the nucleic acid of the invention.
[0082] In one aspect, the polypeptide in the food or nutritional
supplement can be glycosylated. The invention provides edible
enzyme delivery matrices comprising a polypeptide of the invention,
e.g., a polypeptide encoded by the nucleic acid of the invention.
In one aspect, the delivery matrix comprises a pellet. In one
aspect, the polypeptide can be glycosylated. In one aspect, the
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme activity is
thermotolerant. In another aspect, the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity is thermostable.
[0083] The invention provides a food, a feed or a nutritional
supplement comprising a polypeptide of the invention. The invention
provides methods for utilizing an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme as a nutritional supplement in an
animal diet, the method comprising: preparing a nutritional
supplement containing an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
comprising at least thirty contiguous amino acids of a polypeptide
of the invention; and administering the nutritional supplement to
an animal. The animal can be a human, a ruminant or a monogastric
animal. The ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme can be
prepared by expression of a polynucleotide encoding the ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme in an organism selected from
the group consisting of a bacterium, a yeast, a plant, an insect, a
fungus and an animal. The organism can be selected from the group
consisting of an S. pombe, S. cerevisiae, Pichia pastoris, E. coli,
Streptomyces sp., Bacillus sp. and Lactobacillus sp.
[0084] The invention provides edible enzyme delivery matrix
comprising a thermostable recombinant ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme, e.g., a polypeptide of the
invention. The invention provides methods for delivering an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme supplement to an animal, the
method comprising: preparing an edible enzyme delivery matrix in
the form of pellets comprising a granulate edible carrier and a
thermostable recombinant ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzyme, wherein the pellets readily disperse the ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme contained therein into aqueous
media, and administering the edible enzyme delivery matrix to the
animal. The recombinant ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
can comprise a polypeptide of the invention. The ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme can be glycosylated to provide
thermostability at pelletizing conditions. The delivery matrix can
be formed by pelletizing a mixture comprising a grain germ and an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme. The pelletizing
conditions can include application of steam. The pelletizing
conditions can comprise application of a temperature in excess of
about 80.degree. C. for about 5 minutes and the enzyme retains a
specific activity of at least 350 to about 900 units per milligram
of enzyme.
[0085] In certain aspects, a histidine-, phenylalanine- or
tyrosine-containing compound is contacted a polypeptide of the
invention having an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity at a pH in the range of between about pH 3.0 to 9.0, 10.0,
11.0 or more. In other aspects, a histidine-, phenylalanine- or
tyrosine-containing compound is contacted with the ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme at a temperature of about 55.degree.
C., 60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C.,
80.degree. C., 85.degree. C., 90.degree. C., or more.
[0086] In one aspect, invention provides a pharmaceutical
composition comprising an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme of the invention, or a polypeptide encoded by a
nucleic acid of the invention. In one aspect, the pharmaceutical
composition acts as a digestive aid. The lyase can be formulated as
a tablet, gel, geltab, pill, implant, liquid, spray, powder, food,
feed pellet, as an injectable formulation or as an encapsulated
formulation. In one aspect, the polypeptide has ammonia lyase
activity, or phenylalanine ammonia lyase activity, tyrosine ammonia
lyase activity and/or histidine ammonia lyase activity. The
pharmaceutical composition or dietary supplement can be formulated
for the treatment (amelioration) of phenylketonuria (PKU).
[0087] The polypeptide in the pharmaceutical composition or dietary
supplement can be chemically modified to produce a protected form
that possesses better specific activity, prolonged half-life,
and/or reduced immunogenicity in vivo, e.g., the polypeptide can be
chemically modified by glycosylation, pegylation (modified with
polyethylene glycol (PEG), activated PEG, or equivalent),
encapsulation with liposomes or equivalent, encapsulated in
nanostructures (e.g., nanotubules, nano- or microcapsules), or
combinations thereof, or equivalents thereof, e.g., as described by
Wang (2005) Mol Genet Metab. 86(1-2):134-140. Epub 2005 Jul. 11. In
one aspect, the polypeptide is chemically conjugated with activated
PEG, or, 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine,
e.g., as described by Ikeda (2005) Amino Acids 29(3):283-287. Epub
2005 Jun. 28.
[0088] The invention also provides biocompatible matrices such as
sol-gels encapsulating a polypeptide of the invention for use as
pharmaceutical composition or dietary supplement, e.g., to treat or
ameliorate phenylketonuria (PKU), e.g., including silica-based
(e.g., oxysilane) sol-gel matrices. The invention also provides
nano- or microcapsules comprising a polypeptide of the invention
for use as pharmaceutical composition or dietary supplement, e.g.,
to treat or ameliorate phenylketonuria (PKU).
[0089] The invention also provides matrix stabilized enzyme
crystals comprising a polypeptide of the invention for use as
pharmaceutical composition or dietary supplement, e.g., to treat or
ameliorate phenylketonuria (PKU), e.g., as described in U.S. Patent
App. No. 20020182201; for example, the formulation can be a
cross-linked crystalline enzyme and a polymer with a reactive
moiety effective to adhere to the crystal layer of the crystalline
enzyme. The invention also provides polypeptides of the invention
as polymers in the form of multimerized (e.g., multi-functional)
cross-linking forms; which in one aspect comprise a matrix
stabilized enzyme crystal, e.g., a form resistant to degradation by
proteolytic enzymes; and in alternative aspects, the cross-linking
reagents comprise a dialdehyde cross-linking reagents, as discussed
in detail, below.
[0090] The details of one or more aspects of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the description and drawings, and from the claims.
[0091] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0092] The following drawings are illustrative of aspects of the
invention and are not meant to limit the scope of the invention as
encompassed by the claims.
[0093] FIG. 1 is a block diagram of a computer system.
[0094] FIG. 2 is a flow diagram illustrating one aspect of a
process for comparing a new nucleotide or protein sequence with a
database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database.
[0095] FIG. 3 is a flow diagram illustrating one aspect of a
process in a computer for determining whether two sequences are
homologous.
[0096] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence.
[0097] FIG. 5 is an illustration of an exemplary reaction catalyzed
by exemplary phenylalanine ammonia lyases (PALs) of the invention,
wherein phenylalanine is deaminated to trans-cinnamic acid and
ammonia.
[0098] FIGS. 6A and 6B illustrate exemplary catalytic mechanisms of
phenylalanine ammonia lyases (PALs).
[0099] FIG. 7 is an illustration of an exemplary reaction of the
invention, wherein .beta.-Amino Acids are synthesized by
phenylalanine ammonia lyases, or PALs, of the invention.
[0100] FIG. 8 is a table (Table 1), which sets forth exemplary
functions and other information regarding exemplary sequences of
the invention, as discussed below.
[0101] FIGS. 9a, 9b and 9c are a table (Table 2), which sets
information regarding exemplary enzymes of the invention, as
discussed below.
[0102] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0103] The invention provides polypeptides and peptides having at
least one ammonia lyase activity, e.g., at least one phenylalanine
ammonia lyase (PAL), tyrosine ammonia lyase (TAL) and/or histidine
ammonia lyase (HAL) activity, and polynucleotides encoding them,
and methods of making and using these polynucleotides and
polypeptides. The invention also provides ammonia lyase enzymes,
e.g., phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase
(TAL) and histidine ammonia lyase (HAL) enzymes, polynucleotides
encoding these enzymes, the use of such polynucleotides and
polypeptides.
[0104] A number of aspects have been described above and are
described in more detail infra. The embodiments of the invention
include one or more of the described aspects.
[0105] The following explanations of terms and methods are provided
to better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. As used herein, "including" means "comprising." In
addition, the singular forms "a" or "an" or "the" include plural
references unless the context clearly dictates otherwise. For
example, reference to "comprising a protein" includes one or a
plurality of such proteins, and reference to "comprising the cell"
includes reference to one or more cells and equivalents thereof
known to those skilled in the art, and so forth. The term "about"
encompasses the range of experimental error that occurs in any
measurement. Unless otherwise stated, all measurement numbers are
presumed to have the word "about" in front of them even if the word
"about" is not expressly used.
[0106] The invention provides novel phenylalanine ammonia lyase,
tyrosine ammonia lyase and histidine ammonia lyase enzymes. The
invention also provides novel activity assignment for several
previously described putative histidine ammonia lyases (HALs).
Specifically, these putative HALs either have no HAL activity, but
have phenylalanine ammonia lyase (PAL) and/or TAL activity or these
putative HALs additionally have PAL and/or tyrosine ammonia lyase
(TAL) activity.
[0107] Table 1, Table 2 (FIG. 8 and FIG. 9, respectively) and Table
3 (below) detail exemplary activities of polypeptides of the
invention; noting that each polypeptide of the invention can have
more than one specific enzymatic activity. The activities/functions
of exemplary polypeptides of the invention were determined by
sequence comparison (BLAST) analysis with public sequence
databases, such as the NR database available through GenBank and
the Geneseq database available from Thomson Scientific, as
summarized in FIG. 8 and FIG. 9, Tables 1 and 2, respectively.
Table 1, Table 2 (FIG. 8 and FIG. 9, respectively) and Table 3
(below) describe the source organism of the closest hit polypeptide
(see "NR Description" and "NR Organism" columns); the GenBank
accession number of the top BLAST hit for DNA and protein, the
percent sequence identity between the sequence of the invention and
the top BLAST hit, and other descriptions for that particular
exemplary polynucleotide/polypeptide entry and the BLAST
analysis.
[0108] For example, as an aid in reading Table 1, Table 2 (FIG. 8
and FIG. 9, respectively) and Table 3 (below), the polypeptide SEQ
ID NO:2, encoded, e.g., by SEQ ID NO:1, has at least an histidine
ammonia-lyase activity (having an ammonia-lyase enzyme class of
activity), and its activity was determined by a closest BLAST hit
from a sequence initially isolated from Vibrio vulnificus strain
YJO16; Geneseq Protein Accession Code ADS24623; Geneseq DNA
Accession Code ADS61669; or, reading further down the table: the
polypeptide SEQ ID NO:4, encoded, e.g., by SEQ ID NO:3, has at
least a phenylalanine/histidine ammonia-lyase activity (having an
ammonia-lyase enzyme class of activity), and its activity was
determined by a closest BLAST hit from a sequence initially
isolated from Pseudomonas fluorescens PfO-1.
[0109] In one aspect, the invention provides methods for the
synthesis or manufacture of L- and D-phenylalanine and L- and
D-tyrosine as well as L- and D-phenylalanine and L- and D-tyrosine
derivatives (see FIG. 5). In another aspect, the invention provides
methods for the synthesis or manufacture of cinnamic acid and
cinnamic acid derivatives. In yet another aspect, the invention
provides methods for the synthesis or manufacture of
para-hydroxycinnamic acid and para-hydroxyl styrene via
biocatalytic and fermentation. In another aspect, the invention
provides methods for the synthesis or manufacture of ortho-bromo
and ortho-chloro L-phenylalanine and of ortho-bromo and
ortho-chloro D-phenylalanine, as well as derivatives thereof. In
yet another aspect, the invention provides methods for the
synthesis or manufacture of L- and D-.beta.-amino acids (see FIG.
7) and L- and D-histidine and derivatives. In another aspect, the
invention provides methods for the synthesis or manufacture of
urocanoic acid and urocanoic acid derivatives, from histidine and
histidine derivatives.
[0110] In further aspects, the invention provides methods for the
manufacture of bulk and fine chemicals for industrial, medicinal
and agricultural use, using the enzymes of the invention. In other
aspects, the invention provides methods of application of the
enzymes of the invention for enzyme substitution therapy, e.g.,
using PALs for the treatment of phenylketonuria (PKU), an inherited
metabolic disease caused by a deficiency of the enzyme
phenylalanine hydroxylase.
[0111] In one aspect the invention provides compositions (e.g.,
feeds, drugs, dietary supplements) comprising the enzymes,
polypeptides or polynucleotides of the invention. These
compositions can be formulated in a variety of forms, e.g., as
liquids, sprays, films, micelles, liposomes, powders, food, feed
pellets or encapsulated forms, including encapsulated forms.
[0112] Assays for measuring ammonia lyase activity, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase activity, e.g., for determining if a
polypeptide has lyase activity, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase activity, are
well known in the art and are within the scope of the invention;
see, e.g., the PAL enzyme activity assay described in Baedeker
& Schulz (Eur. J. Biochem 2002, 269, 1790-1797), the PAL enzyme
activity assay described in Rother & Retey (Eur. J. Biochem,
2002, 269, 3065-3075), the PAL enzyme activity assay described in
Kyndt et al. (FEBS Letters 2002, 512, 240-24), or the TAL enzyme
activity assay described in Kyndt et al. (FEBS Letters 2002, 512,
240-24).
[0113] The pH of reaction conditions utilized by the invention is
another variable parameter for which the invention provides. In
certain aspects, the pH of the reaction is conducted in the range
of about 3.0 to about 9.0. In other aspects, the pH is about 4.5 or
the pH is about 7.5 or the pH is about 9. Reaction conditions
conducted under alkaline conditions are particularly
advantageous.
[0114] The invention provides for ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase polypeptides of the invention in a variety
of forms and formulations. In the methods of the invention, ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase polypeptides of the invention are
used in a variety of forms and formulations. For example, purified
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase polypeptides can be used in
enzyme substitution therapy, e.g., using PALs for the treatment of
phenylketonuria (PKU), an inherited metabolic disease caused by a
deficiency of the enzyme phenylalanine hydroxylase.
[0115] Alternatively, the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase polypeptide can be expressed in a microorganism using
procedures known in the art. In other aspects, the ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase polypeptides of the invention can be
immobilized on a solid support prior to use in the methods of the
invention. Methods for immobilizing enzymes on solid supports are
commonly known in the art, for example J. Mol. Cat. B: Enzymatic 6
(1999) 29-39; Chivata et al. Biocatalysis: Immobilized cells and
enzymes, J. Mol. Cat. 37 (1986) 1-24: Sharma et al., Immobilized
Biomaterials Techniques and Applications, Angew. Chem. Int. Ed.
Engl. 21 (1982) 837-54: Laskin (Ed.), Enzymes and Immobilized Cells
in Biotechnology.
Nucleic Acids
[0116] In one aspect, the invention provides isolated, recombinant
and synthetic nucleic acids having a sequence identity to an
exemplary sequence of the invention (e.g., any of the odd numbered
SEQ ID NO:s between SEQ ID NO:1 and SEQ ID NO:101; nucleic acids
encoding polypeptides of the invention, e.g., exemplary
polypeptides of the invention, including all even numbered SEQ ID
NO:s between SEQ ID NO:2 and SEQ ID NO:102) including expression
cassettes such as expression vectors, encoding the polypeptides of
the invention. The invention also includes methods for discovering
new ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase polypeptide sequences
using the nucleic acids of the invention. The invention also
includes methods for inhibiting the expression of ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme genes, transcripts and polypeptides
using the nucleic acids of the invention. Also provided are methods
for modifying the nucleic acids of the invention by, e.g.,
synthetic ligation reassembly, optimized directed evolution system
and/or saturation mutagenesis.
[0117] The nucleic acids of the invention can be made, isolated
and/or manipulated by, e.g., cloning and expression of cDNA
libraries, amplification of message or genomic DNA by PCR, and the
like. For example, exemplary sequences of the invention were
initially derived from environmental sources. Regarding the term
"derived" for purposes of the specification and claims, in some
aspects, a substance is "derived" from an organism or source if any
one or more of the following are true: 1) the substance is present
in the organism/source; 2) the substance is removed from the native
host; or, 3) the substance is removed from the native host and is
evolved, for example, by mutagenesis.
[0118] The phrases "nucleic acid" or "nucleic acid sequence" as
used herein refer to an oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense
(complementary) strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material, natural or synthetic in origin. The
phrases "nucleic acid" or "nucleic acid sequence" includes
oligonucleotide, nucleotide, polynucleotide, or to a fragment of
any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense strand, to
peptide nucleic acid (PNA), or to any DNA-like or RNA-like
material, natural or synthetic in origin, including, e.g., iRNA,
ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g.,
iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides,
containing known analogues of natural nucleotides. The term also
encompasses nucleic-acid-like structures with synthetic backbones,
see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;
Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)
Antisense Nucleic Acid Drug Dev 6:153-156. "Oligonucleotide"
includes either a single stranded polydeoxynucleotide or two
complementary polydeoxynucleotide strands which may be chemically
synthesized. Such synthetic oligonucleotides have no 5' phosphate
and thus will not ligate to another oligonucleotide without adding
a phosphate with an ATP in the presence of a kinase. A synthetic
oligonucleotide can ligate to a fragment that has not been
dephosphorylated.
[0119] A "coding sequence of" or a "nucleotide sequence encoding" a
particular polypeptide or protein, is a nucleic acid sequence which
is transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences. The
term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the
coding region (leader and trailer) as well as, where applicable,
intervening sequences (introns) between individual coding segments
(exons). "Operably linked" as used herein refers to a functional
relationship between two or more nucleic acid (e.g., DNA) segments.
Typically, it refers to the functional relationship of
transcriptional regulatory sequence to a transcribed sequence. For
example, a promoter is operably linked to a coding sequence, such
as a nucleic acid of the invention, if it stimulates or modulates
the transcription of the coding sequence in an appropriate host
cell or other expression system. Generally, promoter
transcriptional regulatory sequences that are operably linked to a
transcribed sequence are physically contiguous to the transcribed
sequence, i.e., they are cis-acting. However, some transcriptional
regulatory sequences, such as enhancers, need not be physically
contiguous or located in close proximity to the coding sequences
whose transcription they enhance.
[0120] In one aspect, the invention provides ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme-encoding nucleic acids, and the
polypeptides encoded by them, with a common novelty in that they
are derived from a common source, e.g., an environmental or a
bacterial source.
[0121] In practicing the methods of the invention, homologous genes
can be modified by manipulating a template nucleic acid, as
described herein. The invention can be practiced in conjunction
with any method or protocol or device known in the art, which are
well described in the scientific and patent literature.
[0122] One aspect of the invention is an isolated nucleic acid
comprising one of the sequences of the invention, or a fragment
comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,
200, 300, 400, or 500 or more consecutive bases of a nucleic acid
of the invention. The isolated, nucleic acids may comprise DNA,
including cDNA, genomic DNA and synthetic DNA. The DNA may be
double-stranded or single-stranded and if single stranded may be
the coding strand or non-coding (anti-sense) strand. Alternatively,
the isolated nucleic acids may comprise RNA.
[0123] The isolated nucleic acids of the invention may be used to
prepare one of the polypeptides of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 or more consecutive amino acids of one of the polypeptides of
the invention. Accordingly, another aspect of the invention is an
isolated nucleic acid which encodes one of the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 or more consecutive amino acids of one
of the polypeptides of the invention. The coding sequences of these
nucleic acids may be identical to one of the coding sequences of
one of the nucleic acids of the invention or may be different
coding sequences which encode one of the of the invention having at
least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more
consecutive amino acids of one of the polypeptides of the
invention, as a result of the redundancy or degeneracy of the
genetic code. The genetic code is well known to those of skill in
the art and can be obtained, e.g., on page 214 of B. Lewin, Genes
VI, Oxford University Press, 1997.
[0124] The isolated nucleic acid which encodes one of the
polypeptides of the invention, but is not limited to: only the
coding sequence of a nucleic acid of the invention and additional
coding sequences, such as leader sequences or proprotein sequences
and non-coding sequences, such as introns or non-coding sequences
5' and/or 3' of the coding sequence. Thus, as used herein, the term
"polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only the coding sequence for the
polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequence.
[0125] Alternatively, the nucleic acid sequences of the invention,
may be mutagenized using conventional techniques, such as site
directed mutagenesis, or other techniques familiar to those skilled
in the art, to introduce silent changes into the polynucleotides o
of the invention. As used herein, "silent changes" include, for
example, changes which do not alter the amino acid sequence encoded
by the polynucleotide. Such changes may be desirable in order to
increase the level of the polypeptide produced by host cells
containing a vector encoding the polypeptide by introducing codons
or codon pairs which occur frequently in the host organism.
[0126] The invention also relates to polynucleotides which have
nucleotide changes which result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptides
of the invention. Such nucleotide changes may be introduced using
techniques such as site directed mutagenesis, random chemical
mutagenesis, exonuclease III deletion and other recombinant DNA
techniques. Alternatively, such nucleotide changes may be naturally
occurring allelic variants which are isolated by identifying
nucleic acids which specifically hybridize to probes comprising at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 consecutive bases of one of the sequences of the invention
(or the sequences complementary thereto) under conditions of high,
moderate, or low stringency as provided herein.
[0127] The term "variant" refers to polynucleotides or polypeptides
of the invention modified at one or more base pairs, codons,
introns, exons, or amino acid residues (respectively) yet still
retain the biological activity of an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase of the invention. Variants can be produced
by any number of means included methods such as, for example,
error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,
assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis, exponential ensemble
mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and
any combination thereof.
[0128] General Techniques and Terms
[0129] The nucleic acids used to practice this invention, whether
RNA, siRNA, miRNA, antisense nucleic acid, cDNA, genomic DNA,
vectors, viruses or hybrids thereof, may be isolated from a variety
of sources, genetically engineered, amplified, and/or
expressed/generated recombinantly. Recombinant polypeptides (e.g.,
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes) generated from these
nucleic acids can be individually isolated or cloned and tested for
a desired activity.
[0130] Any recombinant expression system can be used, including
bacterial, mammalian, fungal, yeast, insect or plant cell
expression systems. "Recombinant" polypeptides or proteins refer to
polypeptides or proteins produced by recombinant DNA techniques;
i.e., produced from cells transformed by an exogenous DNA construct
encoding the desired polypeptide or protein. "Synthetic"
polypeptides or protein are those prepared by chemical synthesis.
Solid-phase chemical peptide synthesis methods can also be used to
synthesize the polypeptide or fragments of the invention. Such
method have been known in the art since the early 1960's
(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See
also Stewart, J. M. and Young, J. D., Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., pp.
11-12)) and have recently been employed in commercially available
laboratory peptide design and synthesis kits (Cambridge Research
Biochemicals). Such commercially available laboratory kits have
generally utilized the teachings of H. M. Geysen et al, Proc. Natl.
Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of "rods" or "pins" all of
which are connected to a single plate. Additionally, as used
herein, the term "recombinant" means that the nucleic acid is
adjacent to a "backbone" nucleic acid to which it is not adjacent
in its natural environment.
[0131] Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques, as described in,
e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997)
Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.
Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang
(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;
Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
[0132] Techniques for the manipulation of nucleic acids, such as,
e.g., subcloning, labeling probes (e.g., random-primer labeling
using Klenow polymerase, nick translation, amplification),
sequencing, hybridization and the like are well described in the
scientific and patent literature, see, e.g., Sambrook, ed.,
MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);
LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic
Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0133] Another useful means of obtaining and manipulating nucleic
acids used to practice the methods of the invention is to clone
from genomic samples, and, if desired, screen and re-clone inserts
isolated or amplified from, e.g., genomic clones or cDNA clones.
Sources of nucleic acid used in the methods of the invention
include genomic or cDNA libraries contained in, e.g., mammalian
artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118;
6,025,155; human artificial chromosomes, see, e.g., Rosenfeld
(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);
bacterial artificial chromosomes (BAC); P1 artificial chromosomes,
see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors
(PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids,
recombinant viruses, phages or plasmids.
[0134] In one aspect, a nucleic acid encoding a polypeptide of the
invention is assembled in appropriate phase with a leader sequence
capable of directing secretion of the translated polypeptide or
fragment thereof.
[0135] The invention provides fusion proteins and nucleic acids
encoding them. A polypeptide of the invention can be fused to a
heterologous peptide or polypeptide, such as N-terminal
identification peptides which impart desired characteristics, such
as increased stability or simplified purification. Peptides and
polypeptides of the invention can also be synthesized and expressed
as fusion proteins with one or more additional domains linked
thereto for, e.g., producing a more immunogenic peptide, to more
readily isolate a recombinantly synthesized peptide, to identify
and isolate antibodies and antibody-expressing B cells, and the
like. Detection and purification facilitating domains include,
e.g., metal chelating peptides such as polyhistidine tracts and
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequences such as
Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a
purification domain and the motif-comprising peptide or polypeptide
to facilitate purification. For example, an expression vector can
include an epitope-encoding nucleic acid sequence linked to six
histidine residues followed by a thioredoxin and an enterokinase
cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797;
Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine
residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
epitope from the remainder of the fusion protein. Technology
pertaining to vectors encoding fusion proteins and application of
fusion proteins are well described in the scientific and patent
literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
[0136] The term "isolated" as used herein refers to any substance
removed from its native host; the substance need not be purified.
For example "isolated nucleic acid" refers to a naturally-occurring
nucleic acid that is not immediately contiguous with both of the
sequences with which it is immediately contiguous (one on the 5'
end and one on the 3' end) in the naturally-occurring genome of the
organism from which it is derived. For example, an isolated nucleic
acid can be, without limitation, a recombinant DNA molecule of any
length, provided one of the nucleic acid sequences normally found
immediately flanking that recombinant DNA molecule in a
naturally-occurring genome is removed or absent. Thus, an isolated
nucleic acid includes, without limitation, a recombinant DNA that
exists as a separate molecule (e.g., a cDNA or a genomic DNA
fragment produced by PCR or restriction endonuclease treatment)
independent of other sequences as well as recombinant DNA that is
incorporated into a vector, an autonomously replicating plasmid, a
virus (e.g., a retrovirus, adenovirus, or herpes virus), or into
the genomic DNA of a prokaryote or eukaryote. In addition, an
isolated nucleic acid can include a recombinant DNA molecule that
is part of a hybrid or fusion nucleic acid sequence.
[0137] In one aspect, the term "isolated" means that the material
(e.g., a protein or nucleic acid of the invention) is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a composition
and still be isolated in that such vector or composition is not
part of its natural environment.
[0138] In one aspect, the term "isolated" as used with reference to
nucleic acids also can include any non-naturally-occurring nucleic
acid since non-naturally-occurring nucleic acid sequences are not
found in nature and do not have immediately contiguous sequences in
a naturally-occurring genome. For example, non-naturally-occurring
nucleic acid such as an engineered nucleic acid is considered to be
isolated nucleic acid. Engineered nucleic acid can be made using
common molecular cloning or chemical nucleic acid synthesis
techniques. Isolated non-naturally-occurring nucleic acid can be
independent of other sequences, or incorporated into a vector, an
autonomously replicating plasmid, a virus (e.g., a retrovirus,
adenovirus, or herpes virus), or the genomic DNA of a prokaryote or
eukaryote. In addition, a non-naturally-occurring nucleic acid can
include a nucleic acid molecule that is part of a hybrid or fusion
nucleic acid sequence.
[0139] Purified: The term "purified" as used herein does not
require absolute purity, but rather is intended as a relative term.
Thus, for example, a purified polypeptide or nucleic acid
preparation can be one in which the subject polypeptide or nucleic
acid is at a higher concentration than the polypeptide or nucleic
acid would be in its natural environment within an organism or at a
higher concentration than in the environment from which it was
removed. Individual nucleic acids obtained from a library have been
conventionally purified to electrophoretic homogeneity. The
sequences obtained from these clones could not be obtained directly
either from the library or from total human DNA. The purified
nucleic acids of the invention have been purified from the
remainder of the genomic DNA in the organism by at least
10.sup.4-10.sup.6 fold. In one aspect, the term "purified" includes
nucleic acids which have been purified from the remainder of the
genomic DNA or from other sequences in a library or other
environment by at least one order of magnitude, e.g., in one
aspect, two or three orders, or, four or five orders of
magnitude.
[0140] Enriched: In one aspect, to be "enriched" a nucleic acid
will represent 5% or more of the number of nucleic acid inserts in
a population of nucleic acid backbone molecules. Backbone molecules
according to the invention include nucleic acids such as expression
vectors, self-replicating nucleic acids, viruses, integrating
nucleic acids and other vectors or nucleic acids used to maintain
or manipulate a nucleic acid insert of interest. Typically, the
enriched nucleic acids represent 15% or more of the number of
nucleic acid inserts in the population of recombinant backbone
molecules. More typically, the enriched nucleic acids represent 50%
or more of the number of nucleic acid inserts in the population of
recombinant backbone molecules. In a one aspect, the enriched
nucleic acids represent 90% or more of the number of nucleic acid
inserts in the population of recombinant backbone molecules.
[0141] In one aspect, "amino acid" or "amino acid sequence" as used
herein refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or to a fragment, portion, or subunit of any of these and
to naturally occurring or synthetic molecules. "Amino acid" or
"amino acid sequence" include an oligopeptide, peptide,
polypeptide, or protein sequence, or to a fragment, portion, or
subunit of any of these, and to naturally occurring or synthetic
molecules. The term "polypeptide" as used herein, refers to amino
acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres and may contain modified amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as post-translational
processing, or by chemical modification techniques which are well
known in the art. Modifications can occur anywhere in the
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of a phosphatidylinositol,
cross-linking cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristolyation, oxidation, pegylation, glucan hydrolase processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation and transfer-RNA mediated addition of amino acids to
protein such as arginylation. (See Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W.H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983)). The peptides and polypeptides of the
invention also include all "mimetic" and "peptidomimetic" forms, as
described in further detail, below.
[0142] As used herein, the term "recombinant" means that the
nucleic acid is adjacent to a "backbone" nucleic acid to which it
is not adjacent in its natural environment. Additionally, to be
"enriched" the nucleic acids will represent 5% or more of the
number of nucleic acid inserts in a population of nucleic acid
backbone molecules. Backbone molecules according to the invention
include nucleic acids such as expression vectors, self-replicating
nucleic acids, viruses, integrating nucleic acids and other vectors
or nucleic acids used to maintain or manipulate a nucleic acid
insert of interest. Typically, the enriched nucleic acids represent
15% or more of the number of nucleic acid inserts in the population
of recombinant backbone molecules. More typically, the enriched
nucleic acids represent 50% or more of the number of nucleic acid
inserts in the population of recombinant backbone molecules. In a
one aspect, the enriched nucleic acids represent 90% or more of the
number of nucleic acid inserts in the population of recombinant
backbone molecules.
[0143] The term "saturation mutagenesis", Gene Site Saturation
Mutagenesis, or "GSSM" includes a method that uses degenerate
oligonucleotide primers to introduce point mutations into a
polynucleotide, as described in detail, below.
[0144] The term "optimized directed evolution system" or "optimized
directed evolution" includes a method for reassembling fragments of
related nucleic acid sequences, e.g., related genes, and explained
in detail, below.
[0145] The term "synthetic ligation reassembly" or "SLR" includes a
method of ligating oligonucleotide fragments in a non-stochastic
fashion, and explained in detail, below.
[0146] Transcriptional and Translational Control Sequences
[0147] The invention provides nucleic acid (e.g., DNA) sequences of
the invention operatively linked to expression (e.g.,
transcriptional or translational) control sequence(s), e.g.,
promoters or enhancers, to direct or modulate RNA
synthesis/expression. The expression control sequence can be in an
expression vector. Exemplary bacterial promoters include lacI,
lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic
promoters include CMV immediate early, HSV thymidine kinase, early
and late SV40, LTRs from retrovirus, and mouse metallothionein
I.
[0148] Promoters suitable for expressing a polypeptide in bacteria
include the E. coli lac or trp promoters, the lad promoter, the
lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter,
the lambda PR promoter, the lambda PL promoter, promoters from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), and the acid phosphatase promoter. Eukaryotic
promoters include the CMV immediate early promoter, the HSV
thymidine kinase promoter, heat shock promoters, the early and late
SV40 promoter, LTRs from retroviruses, and the mouse
metallothionein-I promoter. Other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses may also be used. Promoters suitable for expressing the
polypeptide or fragment thereof in bacteria include the E. coli lac
or trp promoters, the lacI promoter, the lacZ promoter, the T3
promoter, the T7 promoter, the gpt promoter, the lambda P.sub.R
promoter, the lambda P.sub.L promoter, promoters from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK)
and the acid phosphatase promoter. Fungal promoters include the
.alpha.-factor promoter. Eukaryotic promoters include the CMV
immediate early promoter, the HSV thymidine kinase promoter, heat
shock promoters, the early and late SV40 promoter, LTRs from
retroviruses and the mouse metallothionein-I promoter. Other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses may also be used.
[0149] As used herein, the term "promoter" includes all sequences
capable of driving transcription of a coding sequence in a cell,
e.g., a plant cell. Thus, promoters used in the constructs of the
invention include cis-acting transcriptional control elements and
regulatory sequences that are involved in regulating or modulating
the timing and/or rate of transcription of a gene. For example, a
promoter can be a cis-acting transcriptional control element,
including an enhancer, a promoter, a transcription terminator, an
origin of replication, a chromosomal integration sequence, 5' and
3' untranslated regions, or an intronic sequence, which are
involved in transcriptional regulation. These cis-acting sequences
typically interact with proteins or other biomolecules to carry out
(turn on/off, regulate, modulate, etc.) transcription.
"Constitutive" promoters are those that drive expression
continuously under most environmental conditions and states of
development or cell differentiation. "Inducible" or "regulatable"
promoters direct expression of the nucleic acid of the invention
under the influence of environmental conditions or developmental
conditions. Examples of environmental conditions that may affect
transcription by inducible promoters include anaerobic conditions,
elevated temperature, drought, or the presence of light.
[0150] "Tissue-specific" promoters are transcriptional control
elements that are only active in particular cells or tissues or
organs, e.g., in plants or animals. Tissue-specific regulation may
be achieved by certain intrinsic factors which ensure that genes
encoding proteins specific to a given tissue are expressed. Such
factors are known to exist in mammals and plants so as to allow for
specific tissues to develop.
[0151] Tissue-Specific Plant Promoters
[0152] The invention provides expression cassettes that can be
expressed in a tissue-specific manner, e.g., that can express an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme of the invention in a
tissue-specific manner. The invention also provides plants or seeds
that express an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme of the
invention in a tissue-specific manner. The tissue-specificity can
be seed specific, stem specific, leaf specific, root specific,
fruit specific and the like.
[0153] The term "plant" includes whole plants, plant parts (e.g.,
leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and
plant cells and progeny of same. The class of plants which can be
used in the method of the invention is generally as broad as the
class of higher plants amenable to transformation techniques,
including angiosperms (monocotyledonous and dicotyledonous plants),
as well as gymnosperms. It includes plants of a variety of ploidy
levels, including polyploid, diploid, haploid and hemizygous
states. As used herein, the term "transgenic plant" includes plants
or plant cells into which a heterologous nucleic acid sequence has
been inserted, e.g., the nucleic acids and various recombinant
constructs (e.g., expression cassettes) of the invention.
[0154] In one aspect, a constitutive promoter such as the CaMV 35S
promoter can be used for expression in specific parts of the plant
or seed or throughout the plant. For example, for overexpression, a
plant promoter fragment can be employed which will direct
expression of a nucleic acid in some or all tissues of a plant,
e.g., a regenerated plant. Such promoters are referred to herein as
"constitutive" promoters and are active under most environmental
conditions and states of development or cell differentiation.
Examples of constitutive promoters include the cauliflower mosaic
virus (CaMV) 35S transcription initiation region, the 1'- or
2'-promoter derived from T-DNA of Agrobacterium tumefaciens, and
other transcription initiation regions from various plant genes
known to those of skill. Such genes include, e.g., ACT11 from
Arabidopsis (Huang (1996) Plant Mol. Biol. 33:125-139); Cat3 from
Arabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet.
251:196-203); the gene encoding stearoyl-acyl carrier protein
desaturase from Brassica napus (Genbank No. X74782, Solocombe
(1994) Plant Physiol. 104:1167-1176); GPc1 from maize (GenBank No.
X15596; Martinez (1989) J. Mol. Biol. 208:551-565); the Gpc2 from
maize (GenBank No. U45855, Manjunath (1997) Plant Mol. Biol.
33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;
5,633,440.
[0155] The invention uses tissue-specific or constitutive promoters
derived from viruses which can include, e.g., the tobamovirus
subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA
92:1679-1683; the rice tungro bacilliform virus (RTBV), which
replicates only in phloem cells in infected rice plants, with its
promoter which drives strong phloem-specific reporter gene
expression; the cassava vein mosaic virus (CVMV) promoter, with
highest activity in vascular elements, in leaf mesophyll cells, and
in root tips (Verdaguer (1996) Plant Mol. Biol. 31:1129-1139).
[0156] Alternatively, the plant promoter may direct expression of
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme-expressing nucleic acid
in a specific tissue, organ or cell type (i.e. tissue-specific
promoters) or may be otherwise under more precise environmental or
developmental control or under the control of an inducible
promoter. Examples of environmental conditions that may affect
transcription include anaerobic conditions, elevated temperature,
the presence of light, or sprayed with chemicals/hormones. For
example, the invention incorporates the drought-inducible promoter
of maize (Busk (1997) supra); the cold, drought, and high salt
inducible promoter from potato (Kirch (1997) Plant Mol. Biol.
33:897 909).
[0157] Tissue-specific promoters can promote transcription only
within a certain time frame of developmental stage within that
tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800,
characterizing the Arabidopsis LEAFY gene promoter. See also Cardon
(1997) Plant J 12:367-77, describing the transcription factor SPL3,
which recognizes a conserved sequence motif in the promoter region
of the A. thaliana floral meristem identity gene AP1; and Mandel
(1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing
the meristem promoter eIF4. Tissue specific promoters which are
active throughout the life cycle of a particular tissue can be
used. In one aspect, the nucleic acids of the invention are
operably linked to a promoter active primarily only in cotton fiber
cells. In one aspect, the nucleic acids of the invention are
operably linked to a promoter active primarily during the stages of
cotton fiber cell elongation, e.g., as described by Rinehart (1996)
supra. The nucleic acids can be operably linked to the Fbl2A gene
promoter to be preferentially expressed in cotton fiber cells
(Ibid). See also, John (1997) Proc. Natl. Acad. Sci. USA
89:5769-5773; John, et al., U.S. Pat. Nos. 5,608,148 and 5,602,321,
describing cotton fiber-specific promoters and methods for the
construction of transgenic cotton plants. Root-specific promoters
may also be used to express the nucleic acids of the invention.
Examples of root-specific promoters include the promoter from the
alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.
123:39-60). Other promoters that can be used to express the nucleic
acids of the invention include, e.g., ovule-specific,
embryo-specific, endosperm-specific, integument-specific, seed
coat-specific promoters, or some combination thereof; a
leaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285
1295, describing a leaf-specific promoter in maize); the ORF13
promoter from Agrobacterium rhizogenes (which exhibits high
activity in roots, see, e.g., Hansen (1997) supra); a maize pollen
specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet.
224:161 168); a tomato promoter active during fruit ripening,
senescence and abscission of leaves and, to a lesser extent, of
flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746);
a pistil-specific promoter from the potato SK2 gene (see, e.g.,
Ficker (1997) Plant Mol. Biol. 35:425 431); the Blec4 gene from
pea, which is active in epidermal tissue of vegetative and floral
shoot apices of transgenic alfalfa making it a useful tool to
target the expression of foreign genes to the epidermal layer of
actively growing shoots or fibers; the ovule-specific BEL1 gene
(see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944);
and/or, the promoter in Klee, U.S. Pat. No. 5,589,583, describing a
plant promoter region is capable of conferring high levels of
transcription in meristematic tissue and/or rapidly dividing
cells.
[0158] Alternatively, plant promoters which are inducible upon
exposure to plant hormones, such as auxins, are used to express the
nucleic acids of the invention. For example, the invention can use
the auxin-response elements E1 promoter fragment (AuxREs) in the
soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407);
the auxin-responsive Arabidopsis GST6 promoter (also responsive to
salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10:
955-966); the auxin-inducible parC promoter from tobacco (Sakai
(1996) 37:906-913); a plant biotin response element (Streit (1997)
Mol. Plant. Microbe Interact. 10:933-937); and, the promoter
responsive to the stress hormone abscisic acid (Sheen (1996)
Science 274:1900-1902).
[0159] The nucleic acids of the invention can also be operably
linked to plant promoters which are inducible upon exposure to
chemicals reagents which can be applied to the plant, such as
herbicides or antibiotics. For example, the maize In2-2 promoter,
activated by benzenesulfonamide herbicide safeners, can be used (De
Veylder (1997) Plant Cell Physiol. 38:568-577); application of
different herbicide safeners induces distinct gene expression
patterns, including expression in the root, hydathodes, and the
shoot apical meristem. Coding sequence can be under the control of,
e.g., a tetracycline-inducible promoter, e.g., as described with
transgenic tobacco plants containing the Avena sativa L. (oat)
arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);
or, a salicylic acid-responsive element (Stange (1997) Plant J.
11:1315-1324). Using chemically- (e.g., hormone- or pesticide-)
induced promoters, i.e., promoter responsive to a chemical which
can be applied to the transgenic plant in the field, expression of
a polypeptide of the invention can be induced at a particular stage
of development of the plant. Thus, the invention also provides for
transgenic plants containing an inducible gene encoding for
polypeptides of the invention whose host range is limited to target
plant species, such as corn, rice, barley, wheat, potato or other
crops, inducible at any stage of development of the crop.
[0160] One of skill will recognize that a tissue-specific plant
promoter may drive expression of operably linked sequences in
tissues other than the target tissue. Thus, a tissue-specific
promoter is one that drives expression preferentially in the target
tissue or cell type, but may also lead to some expression in other
tissues as well.
[0161] The nucleic acids of the invention can also be operably
linked to plant promoters which are inducible upon exposure to
chemicals reagents. These reagents include, e.g., herbicides,
synthetic auxins, or antibiotics which can be applied, e.g.,
sprayed, onto transgenic plants. Inducible expression of the
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme-producing nucleic acids
of the invention will allow the grower to select plants with the
optimal ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme expression
and/or activity. The development of plant parts can thus
controlled. In this way the invention provides the means to
facilitate the harvesting of plants and plant parts. For example,
in various embodiments, the maize In2-2 promoter, activated by
benzenesulfonamide herbicide safeners, is used (De Veylder (1997)
Plant Cell Physiol. 38:568-577); application of different herbicide
safeners induces distinct gene expression patterns, including
expression in the root, hydathodes, and the shoot apical meristem.
Coding sequences of the invention are also under the control of a
tetracycline-inducible promoter, e.g., as described with transgenic
tobacco plants containing the Avena sativa L. (oat) arginine
decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a
salicylic acid-responsive element (Stange (1997) Plant J.
11:1315-1324).
[0162] In some aspects, proper polypeptide expression may require
polyadenylation region at the 3'-end of the coding region. The
polyadenylation region can be derived from the natural gene, from a
variety of other plant (or animal or other) genes, or from genes in
the Agrobacterial T-DNA.
[0163] Expression Cassettes, Vectors and Cloning Vehicles
[0164] The invention provides expression cassettes and vectors and
cloning vehicles comprising nucleic acids of the invention, e.g.,
sequences encoding the ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzymes of the invention. Expression vectors and cloning vehicles
of the invention can comprise viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, Aspergillus and yeast). Vectors of the invention can
include chromosomal, non-chromosomal and synthetic DNA sequences.
Large numbers of suitable vectors are known to those of skill in
the art, and are commercially available. Exemplary vectors are
include: bacterial: pQE vectors (Qiagen), pBLUESCRIPT.TM. plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSGS (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used so long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention.
[0165] "Plasmids" can be commercially available, publicly available
on an unrestricted basis, or can be constructed from available
plasmids in accord with published procedures. Equivalent plasmids
to those described herein are known in the art and will be apparent
to the ordinarily skilled artisan.
[0166] The term "expression cassette" as used herein refers to a
nucleotide sequence which is capable of affecting expression of a
structural gene (i.e., a protein coding sequence, such as an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme of the invention) in a
host compatible with such sequences. Expression cassettes include
at least a promoter operably linked with the polypeptide coding
sequence; and, optionally, with other sequences, e.g.,
transcription termination signals. Additional factors necessary or
helpful in effecting expression may also be used, e.g., enhancers,
alpha-factors. Thus, expression cassettes also include plasmids,
expression vectors, recombinant viruses, any form of recombinant
"naked DNA" vector, and the like. A "vector" comprises a nucleic
acid which can infect, transfect, transiently or permanently
transduce a cell. It will be recognized that a vector can be a
naked nucleic acid, or a nucleic acid complexed with protein or
lipid. The vector optionally comprises viral or bacterial nucleic
acids and/or proteins, and/or membranes (e.g., a cell membrane, a
viral lipid envelope, etc.). Vectors include, but are not limited
to replicons (e.g., RNA replicons, bacteriophages) to which
fragments of DNA may be attached and become replicated. Vectors
thus include, but are not limited to RNA, autonomous
self-replicating circular or linear DNA or RNA (e.g., plasmids,
viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and
include both the expression and non-expression plasmids. Where a
recombinant microorganism or cell culture is described as hosting
an "expression vector" this includes both extra-chromosomal
circular and linear DNA and DNA that has been incorporated into the
host chromosome(s). Where a vector is being maintained by a host
cell, the vector may either be stably replicated by the cells
during mitosis as an autonomous structure, or is incorporated
within the host's genome.
[0167] The expression vector can comprise a promoter, a ribosome
binding site for translation initiation and a transcription
terminator. The vector may also include appropriate sequences for
amplifying expression. Mammalian expression vectors can comprise an
origin of replication, any necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In some aspects, DNA sequences derived
from the SV40 splice and polyadenylation sites may be used to
provide the required non-transcribed genetic elements.
[0168] In one aspect, the expression vectors contain one or more
selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli, and the S.
cerevisiae TRP1 gene. Promoter regions can be selected from any
desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0169] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells can also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA that
can be from about 10 to about 300 bp in length. They can act on a
promoter to increase its transcription. Exemplary enhancers include
the SV40 enhancer on the late side of the replication origin by 100
to 270, the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and the
adenovirus enhancers.
[0170] A nucleic acid sequence can be inserted into a vector by a
variety of procedures. In general, the sequence is ligated to the
desired position in the vector following digestion of the insert
and the vector with appropriate restriction endonucleases.
Alternatively, blunt ends in both the insert and the vector may be
ligated. A variety of cloning techniques are known in the art,
e.g., as described in Ausubel and Sambrook. Such procedures and
others are deemed to be within the scope of those skilled in the
art.
[0171] The vector can be in the form of a plasmid, a viral
particle, or a phage. Other vectors include chromosomal,
non-chromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described by, e.g., Sambrook.
[0172] Particular bacterial vectors which can be used include the
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison,
Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBLUESCRIPT
II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a,
pKK223-3, pKK233-3, DR540, pRITS (Pharmacia), pKK232-8 and pCM7.
Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any
other vector may be used as long as it is replicable and viable in
the host cell.
[0173] The nucleic acids of the invention can be expressed in
expression cassettes, vectors or viruses and transiently or stably
expressed in plant cells and seeds. One exemplary transient
expression system uses episomal expression systems, e.g.,
cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus
by transcription of an episomal mini-chromosome containing
supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA
87:1633-1637. Alternatively, coding sequences, i.e., all or
sub-fragments of sequences of the invention can be inserted into a
plant host cell genome becoming an integral part of the host
chromosomal DNA. Sense or antisense transcripts can be expressed in
this manner. A vector comprising the sequences (e.g., promoters or
coding regions) from nucleic acids of the invention can comprise a
marker gene that confers a selectable phenotype on a plant cell or
a seed. For example, the marker may encode biocide resistance,
particularly antibiotic resistance, such as resistance to
kanamycin, G418, bleomycin, hygromycin, or herbicide resistance,
such as resistance to chlorosulfuron or Basta.
[0174] Expression vectors capable of expressing nucleic acids and
proteins in plants are well known in the art, and can include,
e.g., vectors from Agrobacterium spp., potato virus X (see, e.g.,
Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see,
e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see,
e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see,
e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus
(see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476),
cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant.
Microbe Interact. 10:1094-1101), maize Ac/Ds transposable element
(see, e.g., Rubin (1997) Mol. Cell. Biol. 17:6294-6302; Kunze
(1996) Curr. Top. Microbiol. Immunol. 204:161-194), and the maize
suppressor-mutator (Spm) transposable element (see, e.g., Schlappi
(1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.
[0175] In one aspect, the expression vector can have two
replication systems to allow it to be maintained in two organisms,
for example in mammalian or insect cells for expression and in a
prokaryotic host for cloning and amplification. Furthermore, for
integrating expression vectors, the expression vector can contain
at least one sequence homologous to the host cell genome. It can
contain two homologous sequences which flank the expression
construct. The integrating vector can be directed to a specific
locus in the host cell by selecting the appropriate homologous
sequence for inclusion in the vector. Constructs for integrating
vectors are well known in the art.
[0176] Expression vectors of the invention may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed, e.g., genes which render the
bacteria resistant to drugs such as ampicillin, chloramphenicol,
erythromycin, kanamycin, neomycin and tetracycline. Selectable
markers can also include biosynthetic genes, such as those in the
histidine, tryptophan and leucine biosynthetic pathways.
[0177] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lad, lacZ, T3, T'7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using
chloramphenicol transferase (CAT) vectors or other vectors with
selectable markers. In addition, the expression vectors in one
aspect contain one or more selectable marker genes to provide a
phenotypic trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic cell
culture, or such as tetracycline or ampicillin resistance in E.
coli.
[0178] Mammalian expression vectors may also comprise an origin of
replication, any necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences and 5' flanking
nontranscribed sequences. In some aspects, DNA sequences derived
from the SV40 splice and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0179] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells may also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA,
usually from about 10 to about 300 bp in length that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin by 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin and the adenovirus
enhancers.
[0180] In addition, the expression vectors typically contain one or
more selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli and the S.
cerevisiae TRP1 gene.
[0181] In some aspects, the nucleic acid encoding one of the
polypeptides of the invention, or fragments comprising at least
about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof is assembled in appropriate phase
with a leader sequence capable of directing secretion of the
translated polypeptide or fragment thereof. Optionally, the nucleic
acid can encode a fusion polypeptide in which one of the
polypeptides of the invention, or fragments comprising at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino
acids thereof is fused to heterologous peptides or polypeptides,
such as N-terminal identification peptides which impart desired
characteristics, such as increased stability or simplified
purification.
[0182] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is ligated
to the desired position in the vector following digestion of the
insert and the vector with appropriate restriction endonucleases.
Alternatively, blunt ends in both the insert and the vector may be
ligated.
[0183] A variety of cloning techniques are disclosed in Ausubel et
al. Current Protocols in Molecular Biology, John Wiley 503 Sons,
Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory
Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989. Such
procedures and others are deemed to be within the scope of those
skilled in the art.
[0184] The vector may be, for example, in the form of a plasmid, a
viral particle, or a phage. Other vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus and pseudorabies. A variety
of cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are described by Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.,
(1989).
[0185] Host Cells and Transformed Cells
[0186] The invention also provides a transformed cell comprising a
nucleic acid sequence of the invention, e.g., a sequence encoding
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme of the
invention, or a vector of the invention. The host cell may be any
of the host cells familiar to those skilled in the art, including
prokaryotic cells, eukaryotic cells, such as bacterial cells,
fungal cells, yeast cells, mammalian cells, insect cells, or plant
cells. Exemplary bacterial cells include E. coli, Streptomyces,
Bacillus subtilis, Bacillus cereus, Salmonella typhimurium and
various species within the genera Streptomyces and Staphylococcus.
Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
Exemplary animal cells include CHO, COS or Bowes melanoma or any
mouse or human cell line. The selection of an appropriate host is
within the abilities of those skilled in the art. Techniques for
transforming a wide variety of higher plant species are well known
and described in the technical and scientific literature. See,
e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S. Pat. No.
5,750,870.
[0187] The vector can be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0188] In one aspect, the nucleic acids or vectors of the invention
are introduced into the cells for screening, thus, the nucleic
acids enter the cells in a manner suitable for subsequent
expression of the nucleic acid. The method of introduction is
largely dictated by the targeted cell type. Exemplary methods
include CaPO.sub.4 precipitation, liposome fusion, lipofection
(e.g., LIPOFECTIN.TM.), electroporation, viral infection, etc. The
candidate nucleic acids may stably integrate into the genome of the
host cell (for example, with retroviral introduction) or may exist
either transiently or stably in the cytoplasm (i.e. through the use
of traditional plasmids, utilizing standard regulatory sequences,
selection markers, etc.). As many pharmaceutically important
screens require human or model mammalian cell targets, retroviral
vectors capable of transfecting such targets can be used.
[0189] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0190] Cells can be harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0191] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0192] Cell-free translation systems can also be employed to
produce a polypeptide of the invention. Cell-free translation
systems can use mRNAs transcribed from a DNA construct comprising a
promoter operably linked to a nucleic acid encoding the polypeptide
or fragment thereof. In some aspects, the DNA construct may be
linearized prior to conducting an in vitro transcription reaction.
The transcribed mRNA is then incubated with an appropriate
cell-free translation extract, such as a rabbit reticulocyte
extract, to produce the desired polypeptide or fragment
thereof.
[0193] The expression vectors can contain one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0194] Host cells containing the polynucleotides of interest, e.g.,
nucleic acids of the invention, can be cultured in conventional
nutrient media modified as appropriate for activating promoters,
selecting transformants or amplifying genes. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression and will
be apparent to the ordinarily skilled artisan. The clones which are
identified as having the specified enzyme activity may then be
sequenced to identify the polynucleotide sequence encoding an
enzyme having the enhanced activity.
[0195] The invention provides a method for overexpressing a
recombinant ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme in a
cell comprising expressing a vector comprising a nucleic acid of
the invention, e.g., a nucleic acid comprising a nucleic acid
sequence with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more sequence identity to an exemplary
sequence of the invention over a region of at least about 100
residues, wherein the sequence identities are determined by
analysis with a sequence comparison algorithm or by visual
inspection, or, a nucleic acid that hybridizes under stringent
conditions to a nucleic acid sequence of the invention. The
overexpression can be effected by any means, e.g., use of a high
activity promoter, a dicistronic vector or by gene amplification of
the vector.
[0196] The nucleic acids of the invention can be expressed, or
overexpressed, in any in vitro or in vivo expression system. Any
cell culture systems can be employed to express, or over-express,
recombinant protein, including bacterial, insect, yeast, fungal or
mammalian cultures. Over-expression can be effected by appropriate
choice of promoters, enhancers, vectors (e.g., use of replicon
vectors, dicistronic vectors (see, e.g., Gurtu (1996) Biochem.
Biophys. Res. Commun. 229:295-8), media, culture systems and the
like. In one aspect, gene amplification using selection markers,
e.g., glutamine synthetase (see, e.g., Sanders (1987) Dev. Biol.
Stand. 66:55-63), in cell systems are used to overexpress the
polypeptides of the invention.
[0197] The host cell may be any of the host cells familiar to those
skilled in the art, including prokaryotic cells, eukaryotic cells,
mammalian cells, insect cells, or plant cells. As representative
examples of appropriate hosts, there may be mentioned: bacterial
cells, such as E. coli, Streptomyces, Bacillus subtilis, Bacillus
cereus, Salmonella typhimurium and various species within the
genera Streptomyces and Staphylococcus, fungal cells, such as
yeast, insect cells such as Drosophila S2 and Spodoptera Sf9,
animal cells such as CHO, COS or Bowes melanoma and adenoviruses.
The selection of an appropriate host is within the abilities of
those skilled in the art.
[0198] The vector may be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0199] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0200] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0201] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts
(described by Gluzman, Cell, 23:175, 1981) and other cell lines
capable of expressing proteins from a compatible vector, such as
the C127, 3T3, CHO, HeLa and BHK cell lines.
[0202] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0203] Alternatively, the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 or more consecutive amino acids thereof can be
synthetically produced by conventional peptide synthesizers. In
other aspects, fragments or portions of the polypeptides may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, the fragments may be employed as
intermediates for producing the full-length polypeptides.
[0204] Cell-free translation systems can also be employed to
produce one of the polypeptides of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 or more consecutive amino acids thereof using mRNAs transcribed
from a DNA construct comprising a promoter operably linked to a
nucleic acid encoding the polypeptide or fragment thereof. In some
aspects, the DNA construct may be linearized prior to conducting an
in vitro transcription reaction. The transcribed mRNA is then
incubated with an appropriate cell-free translation extract, such
as a rabbit reticulocyte extract, to produce the desired
polypeptide or fragment thereof.
[0205] Amplification of Nucleic Acids
[0206] In practicing the invention, nucleic acids of the invention
and nucleic acids encoding the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes of the invention, or modified nucleic acids of the
invention, can be reproduced by amplification. Amplification can
also be used to clone or modify the nucleic acids of the invention.
Thus, the invention provides amplification primer sequence pairs
for amplifying nucleic acids of the invention, including exemplary
sequences of the invention, e.g., all odd SEQ ID NO:s between SEQ
ID NO:1 and SEQ ID NO:101. One of skill in the art can design
amplification primer sequence pairs for any part of or the full
length of these sequences.
[0207] In one aspect, the invention provides a nucleic acid
amplified by a primer pair of the invention, e.g., a primer pair as
set forth by about the first (the 5') 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 or more residues of a nucleic
acid of the invention, and about the first (the 5') 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of
the complementary strand.
[0208] The invention provides an amplification primer sequence pair
for amplifying a nucleic acid encoding a polypeptide having an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme activity, wherein the
primer pair is capable of amplifying a nucleic acid comprising a
sequence of the invention, or fragments or subsequences thereof.
One or each member of the amplification primer sequence pair can
comprise an oligonucleotide comprising at least about 10 to 50 or
more consecutive bases of the sequence, or about 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more consecutive
bases of the sequence. The invention provides amplification primer
pairs, wherein the primer pair comprises a first member having a
sequence as set forth by about the first (the 5') 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of a
nucleic acid of the invention, and a second member having a
sequence as set forth by about the first (the 5') 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of the
complementary strand of the first member. The invention provides
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
methods of making an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
by amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. In one aspect, the
amplification primer pair amplifies a nucleic acid from a library,
e.g., a gene library, such as an environmental library.
[0209] Amplification reactions can also be used to quantify the
amount of nucleic acid in a sample (such as the amount of message
in a cell sample), label the nucleic acid (e.g., to apply it to an
array or a blot), detect the nucleic acid, or quantify the amount
of a specific nucleic acid in a sample. In one aspect of the
invention, message isolated from a cell or a cDNA library are
amplified.
[0210] The skilled artisan can select and design suitable
oligonucleotide amplification primers. Amplification methods are
also well known in the art, and include, e.g., polymerase chain
reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND
APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR
STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase
chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;
Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);
transcription amplification (see, e.g., Kwoh (1989) Proc. Natl.
Acad. Sci. USA 86:1173); and, self-sustained sequence replication
(see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q
Beta replicase amplification (see, e.g., Smith (1997) J. Clin.
Microbiol. 35:1477-1491), automated Q-beta replicase amplification
assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and
other RNA polymerase mediated techniques (e.g., NASBA, Cangene,
Mississauga, Ontario); see also Berger (1987) Methods Enzymol.
152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and
4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
[0211] Determining the Degree of Sequence Identity
[0212] The invention provides nucleic acids comprising sequences
having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more, or complete (100%) sequence identity (homology)
to an exemplary nucleic acid of the invention (e.g., e.g., all odd
SEQ ID NO:s between SEQ ID NO:1 and SEQ ID NO:101) over a region of
at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,
residues. The invention provides polypeptides comprising sequences
having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more, or complete (100%) sequence identity to an
exemplary polypeptide of the invention (e.g., all even SEQ ID NO:s
between SEQ ID NO:2 and SEQ ID NO:102, and subsequences thereof,
including enzymatically active fragments thereof), and nucleic
acids encoding them (including both strands, i.e., sense and
nonsense, coding or noncoding). The extent of sequence identity
(homology) may be determined using any computer program and
associated parameters, including those described herein, such as
BLAST 2.2.2. or FASTA version 3.0t78, with the default
parameters.
[0213] Nucleic acid sequences of the invention can comprise at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 or more consecutive nucleotides of an exemplary sequence of
the invention and sequences substantially identical thereto.
Homologous sequences and fragments of nucleic acid sequences of the
invention can refer to a sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence
identity (homology) to these sequences.
[0214] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides, refers to two or more sequences that
have, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more nucleotide or amino acid residue (sequence)
identity, when compared and aligned for maximum correspondence, as
measured using one of the known sequence comparison algorithms or
by visual inspection. In alternative aspects, the substantial
identity exists over a region of at least about 100 or more
residues and most commonly the sequences are substantially
identical over at least about 150 to 200 or more residues. In some
aspects, the sequences are substantially identical over the entire
length of the coding regions.
[0215] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides, refers to two or more sequences that
have, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more nucleotide or amino acid residue (sequence)
identity, when compared and aligned for maximum correspondence, as
measured using one of the known sequence comparison algorithms or
by visual inspection. In alternative aspects, the substantial
identity exists over a region of at least about 100 or more
residues and most commonly the sequences are substantially
identical over at least about 150 to 200 or more residues. In some
aspects, the sequences are substantially identical over the entire
length of the coding regions.
[0216] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence by one or more
conservative or non-conservative amino acid substitutions,
deletions, or insertions. In one aspect, the substitution occurs at
a site that is not the active site of the molecule, or,
alternatively the substitution occurs at a site that is the active
site of the molecule, provided that the polypeptide essentially
retains its functional (enzymatic) properties. A conservative amino
acid substitution, for example, substitutes one amino acid for
another of the same class (e.g., substitution of one hydrophobic
amino acid, such as isoleucine, valine, leucine, or methionine, for
another, or substitution of one polar amino acid for another, such
as substitution of arginine for lysine, glutamic acid for aspartic
acid or glutamine for asparagine). One or more amino acids can be
deleted, for example, from an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase polypeptide, resulting in modification of the structure of
the polypeptide, without significantly altering its biological
activity. For example, amino- or carboxyl-terminal amino acids that
are not required for ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
biological activity can be removed. Modified polypeptide sequences
of the invention can be assayed for ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme biological activity by any number of
methods, including contacting the modified polypeptide sequence
with a substrate and determining whether the modified polypeptide
decreases the amount of specific substrate in the assay or
increases the bioproducts of the enzymatic reaction of a functional
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase polypeptide with the
substrate.
[0217] Homology (sequence identity) may be determined using any of
the computer programs and parameters described herein, including
FASTA version 3.0t78 with the default parameters. Homologous
sequences also include RNA sequences in which uridines replace the
thymines in the nucleic acid sequences of the invention. The
homologous sequences may be obtained using any of the procedures
described herein or may result from the correction of a sequencing
error. It will be appreciated that the nucleic acid sequences of
the invention can be represented in the traditional single
character format (See the inside back cover of Stryer, Lubert.
Biochemistry, 3rd Ed., W. H Freeman & Co., New York.) or in any
other format which records the identity of the nucleotides in a
sequence.
[0218] As used herein, the terms "computer," "computer program" and
"processor" are used in their broadest general contexts and
incorporate all such devices, as described in detail, below. A
"coding sequence of" or a "sequence encodes" a particular
polypeptide or protein, is a nucleic acid sequence which is
transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences.
[0219] Various sequence comparison programs identified elsewhere in
this patent specification are particularly contemplated for use in
this aspect of the invention. Protein and/or nucleic acid sequence
homologies may be evaluated using any of the variety of sequence
comparison algorithms and programs known in the art. Such
algorithms and programs include, but are by no means limited to,
TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (see, e.g., Pearson and
Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul
et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson Nucleic Acids
Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol.
266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410,
1990; Altschul et al., Nature Genetics 3:266-272, 1993).
[0220] Homology or identity is often measured using sequence
analysis software (e.g., Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705). Such software
matches similar sequences by assigning degrees of homology to
various deletions, substitutions and other modifications. The terms
"homology" and "identity" in the context of two or more nucleic
acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same when compared
and aligned for maximum correspondence over a comparison window or
designated region as measured using any number of sequence
comparison algorithms or by manual alignment and visual
inspection.
[0221] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0222] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequence for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP.TM., BESTFIT.TM., FASTA
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection. Other algorithms for determining
homology or identity include, for example, in addition to a BLAST
program (Basic Local Alignment Search Tool at the National Center
for Biological Information), ALIGN.TM., AMAS (Analysis of Multiply
Aligned Sequences), AMPS (Protein Multiple Sequence Alignment),
ASSET (Aligned Segment Statistical Evaluation Tool), BANDS,
BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node),
BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points,
BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Waterman algorithm, DARWIN.TM., Las Vegas algorithm, FNAT
(Forced Nucleotide Alignment Tool), FRAMEALIGN.TM.,
FRAMESEARCH.TM., DYNAMIC.TM., FILTER.TM., FSAP.TM. (Fristensky
Sequence Analysis Package), GAP (Global Alignment Program),
GENAL.TM., GIBBS.TM., GENQUEST.TM., ISSC.TM. (Sensitive Sequence
Comparison), LALIGN.TM. (Local Sequence Alignment), LCP.TM. (Local
Content Program), MACAW.TM. (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP.TM.,
MBLKN.TM., PIMA.TM. (Pattern-Induced Multi-sequence Alignment),
SAGA.TM. (Sequence Alignment by Genetic Algorithm) and WHAT-IF.TM..
Such alignment programs can also be used to screen genome databases
to identify polynucleotide sequences having substantially identical
sequences. A number of genome databases are available, for example,
a substantial portion of the human genome is available as part of
the Human Genome Sequencing Project (Gibbs, 1995). At least
twenty-one other genomes have already been sequenced, including,
for example, M. genitalium (Fraser et al., 1995), M. jannaschii
(Bult et al., 1996), H. influenzae (Fleischmann et al., 1995), E.
coli (Blattner et al., 1997) and yeast (S. cerevisiae) (Mewes et
al., 1997) and D. melanogaster (Adams et al., 2000). Significant
progress has also been made in sequencing the genomes of model
organism, such as mouse, C. elegans and Arabadopsis sp. Several
databases containing genomic information annotated with some
functional information are maintained by different organizations
and may be accessible via the internet.
[0223] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410,
1990, respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, an expectation (E) of 10,
M=5, N=-4 and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength of 3
and expectations (E) of 10 and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4 and a
comparison of both strands.
[0224] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993). One measure of
similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a references sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more in one aspect less than about 0.01 and
most in one aspect less than about 0.001.
[0225] In one aspect, protein and nucleic acid sequence homologies
are evaluated using the Basic Local Alignment Search Tool ("BLAST")
In particular, five specific BLAST programs are used to perform the
following task: [0226] (1) BLASTP and BLAST3 compare an amino acid
query sequence against a protein sequence database; [0227] (2)
BLASTN compares a nucleotide query sequence against a nucleotide
sequence database; [0228] (3) BLASTX compares the six-frame
conceptual translation products of a query nucleotide sequence
(both strands) against a protein sequence database; [0229] (4)
TBLASTN compares a query protein sequence against a nucleotide
sequence database translated in all six reading frames (both
strands); and [0230] (5) TBLASTX compares the six-frame
translations of a nucleotide query sequence against the six-frame
translations of a nucleotide sequence database.
[0231] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic acid
sequence and a test sequence which is in one aspect obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are in one aspect identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. In one aspect,
the scoring matrix used is the BLOSUM62 matrix (Gonnet (1992)
Science 256:1443-1445; Henikoff and Henikoff (1993) Proteins
17:49-61). Less in one aspect, the PAM or PAM250 matrices may also
be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for
Detecting Distance Relationships: Atlas of Protein Sequence and
Structure, Washington: National Biomedical Research Foundation).
BLAST programs are accessible through the U.S. National Library of
Medicine.
[0232] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In
some aspects, the parameters may be the default parameters used by
the algorithms in the absence of instructions from the user.
[0233] Computer Systems and Computer Program Products
[0234] To determine and identify sequence identities, structural
homologies, motifs and the like in silico, a nucleic acid or
polypeptide sequence of the invention can be stored, recorded, and
manipulated on any medium which can be read and accessed by a
computer.
[0235] Accordingly, the invention provides computers, computer
systems, computer readable mediums, computer programs products and
the like recorded or stored thereon the nucleic acid and
polypeptide sequences of the invention. As used herein, the words
"recorded" and "stored" refer to a process for storing information
on a computer medium. A skilled artisan can readily adopt any known
methods for recording information on a computer readable medium to
generate manufactures comprising one or more of the nucleic acid
and/or polypeptide sequences of the invention.
[0236] The polypeptides of the invention include the polypeptide
sequences of the invention, e.g., the exemplary sequences of the
invention, and sequences substantially identical thereto, and
fragments of any of the preceding sequences. Substantially
identical, or homologous, polypeptide sequences refer to a
polypeptide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity (homology) to an exemplary sequence of the invention.
[0237] Homology (sequence identity) may be determined using any of
the computer programs and parameters described herein. A nucleic
acid or polypeptide sequence of the invention can be stored,
recorded and manipulated on any medium which can be read and
accessed by a computer. As used herein, the words "recorded" and
"stored" refer to a process for storing information on a computer
medium. A skilled artisan can readily adopt any of the presently
known methods for recording information on a computer readable
medium to generate manufactures comprising one or more of the
nucleic acid sequences of the invention, one or more of the
polypeptide sequences of the invention. Another aspect of the
invention is a computer readable medium having recorded thereon at
least 2, 5, 10, 15, or 20 or more nucleic acid or polypeptide
sequences of the invention.
[0238] Another aspect of the invention is a computer readable
medium having recorded thereon one or more of the nucleic acid
sequences of the invention. Another aspect of the invention is a
computer readable medium having recorded thereon one or more of the
polypeptide sequences of the invention. Another aspect of the
invention is a computer readable medium having recorded thereon at
least 2, 5, 10, 15, or 20 or more of the nucleic acid or
polypeptide sequences as set forth above.
[0239] Computer readable media include magnetically readable media,
optically readable media, electronically readable media and
magnetic/optical media. For example, the computer readable media
may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital
Versatile Disk (DVD), Random Access Memory (RAM), or Read Only
Memory (ROM) as well as other types of other media known to those
skilled in the art.
[0240] Aspects of the invention include systems (e.g., internet
based systems), particularly computer systems which store and
manipulate the sequence information described herein. One example
of a computer system 100 is illustrated in block diagram form in
FIG. 1. As used herein, "a computer system" refers to the hardware
components, software components and data storage components used to
analyze a nucleotide sequence of a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention. The computer
system 100 typically includes a processor for processing, accessing
and manipulating the sequence data. The processor 105 can be any
well-known type of central processing unit, such as, for example,
the Pentium III from Intel Corporation, or similar processor from
Sun, Motorola, Compaq, AMD or International Business Machines.
[0241] Typically the computer system 100 is a general purpose
system that comprises the processor 105 and one or more internal
data storage components 110 for storing data and one or more data
retrieving devices for retrieving the data stored on the data
storage components. A skilled artisan can readily appreciate that
any one of the currently available computer systems are
suitable.
[0242] In one particular aspect, the computer system 100 includes a
processor 105 connected to a bus which is connected to a main
memory 115 (in one aspect implemented as RAM) and one or more
internal data storage devices 110, such as a hard drive and/or
other computer readable media having data recorded thereon. In some
aspects, the computer system 100 further includes one or more data
retrieving device 118 for reading the data stored on the internal
data storage devices 110.
[0243] The data retrieving device 118 may represent, for example, a
floppy disk drive, a compact disk drive, a magnetic tape drive, or
a modem capable of connection to a remote data storage system
(e.g., via the internet) etc. In some aspects, the internal data
storage device 110 is a removable computer readable medium such as
a floppy disk, a compact disk, a magnetic tape, etc. containing
control logic and/or data recorded thereon. The computer system 100
may advantageously include or be programmed by appropriate software
for reading the control logic and/or the data from the data storage
component once inserted in the data retrieving device.
[0244] The computer system 100 includes a display 120 which is used
to display output to a computer user. It should also be noted that
the computer system 100 can be linked to other computer systems
125a-c in a network or wide area network to provide centralized
access to the computer system 100.
[0245] Software for accessing and processing the nucleotide
sequences of a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention, (such as search tools,
compare tools and modeling tools etc.) may reside in main memory
115 during execution.
[0246] In some aspects, the computer system 100 may further
comprise a sequence comparison algorithm for comparing a nucleic
acid sequence of the invention, or a polypeptide sequence of the
invention, stored on a computer readable medium to a reference
nucleotide or polypeptide sequence(s) stored on a computer readable
medium. A "sequence comparison algorithm" refers to one or more
programs which are implemented (locally or remotely) on the
computer system 100 to compare a nucleotide sequence with other
nucleotide sequences and/or compounds stored within a data storage
means. For example, the sequence comparison algorithm may compare
the nucleotide sequences of a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention, stored on a
computer readable medium to reference sequences stored on a
computer readable medium to identify homologies or structural
motifs.
[0247] FIG. 2 is a flow diagram illustrating one aspect of a
process 200 for comparing a new nucleotide or protein sequence with
a database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database. The
database of sequences can be a private database stored within the
computer system 100, or a public database such as GENBANK that is
available through the Internet.
[0248] The process 200 begins at a start state 201 and then moves
to a state 202 wherein the new sequence to be compared is stored to
a memory in a computer system 100. As discussed above, the memory
could be any type of memory, including RAM or an internal storage
device.
[0249] The process 200 then moves to a state 204 wherein a database
of sequences is opened for analysis and comparison. The process 200
then moves to a state 206 wherein the first sequence stored in the
database is read into a memory on the computer. A comparison is
then performed at a state 210 to determine if the first sequence is
the same as the second sequence. It is important to note that this
step is not limited to performing an exact comparison between the
new sequence and the first sequence in the database. Well-known
methods are known to those of skill in the art for comparing two
nucleotide or protein sequences, even if they are not identical.
For example, gaps can be introduced into one sequence in order to
raise the homology level between the two tested sequences. The
parameters that control whether gaps or other features are
introduced into a sequence during comparison are normally entered
by the user of the computer system.
[0250] Once a comparison of the two sequences has been performed at
the state 210, a determination is made at a decision state 210
whether the two sequences are the same. Of course, the term "same"
is not limited to sequences that are absolutely identical.
Sequences that are within the homology parameters entered by the
user will be marked as "same" in the process 200.
[0251] If a determination is made that the two sequences are the
same, the process 200 moves to a state 214 wherein the name of the
sequence from the database is displayed to the user. This state
notifies the user that the sequence with the displayed name
fulfills the homology constraints that were entered. Once the name
of the stored sequence is displayed to the user, the process 200
moves to a decision state 218 wherein a determination is made
whether more sequences exist in the database. If no more sequences
exist in the database, then the process 200 terminates at an end
state 220. However, if more sequences do exist in the database,
then the process 200 moves to a state 224 wherein a pointer is
moved to the next sequence in the database so that it can be
compared to the new sequence. In this manner, the new sequence is
aligned and compared with every sequence in the database.
[0252] It should be noted that if a determination had been made at
the decision state 212 that the sequences were not homologous, then
the process 200 would move immediately to the decision state 218 in
order to determine if any other sequences were available in the
database for comparison.
[0253] Accordingly, one aspect of the invention is a computer
system comprising a processor, a data storage device having stored
thereon a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, a data storage device having retrievably
stored thereon reference nucleotide sequences or polypeptide
sequences to be compared to a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention and a
sequence comparer for conducting the comparison. The sequence
comparer may indicate a homology level between the sequences
compared or identify structural motifs in the above described
nucleic acid code a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention, or it may identify
structural motifs in sequences which are compared to these nucleic
acid codes and polypeptide codes. In some aspects, the data storage
device may have stored thereon the sequences of at least 2, 5, 10,
15, 20, 25, 30 or 40 or more of the nucleic acid sequences of the
invention, or the polypeptide sequences of the invention.
[0254] Another aspect of the invention is a method for determining
the level of homology between a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention and a
reference nucleotide sequence. The method including reading the
nucleic acid code or the polypeptide code and the reference
nucleotide or polypeptide sequence through the use of a computer
program which determines homology levels and determining homology
between the nucleic acid code or polypeptide code and the reference
nucleotide or polypeptide sequence with the computer program. The
computer program may be any of a number of computer programs for
determining homology levels, including those specifically
enumerated herein, (e.g., BLAST2N with the default parameters or
with any modified parameters). The method may be implemented using
the computer systems described above. The method may also be
performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 or
more of the above described nucleic acid sequences of the
invention, or the polypeptide sequences of the invention through
use of the computer program and determining homology between the
nucleic acid codes or polypeptide codes and reference nucleotide
sequences or polypeptide sequences.
[0255] FIG. 3 is a flow diagram illustrating one aspect of a
process 250 in a computer for determining whether two sequences are
homologous. The process 250 begins at a start state 252 and then
moves to a state 254 wherein a first sequence to be compared is
stored to a memory. The second sequence to be compared is then
stored to a memory at a state 256. The process 250 then moves to a
state 260 wherein the first character in the first sequence is read
and then to a state 262 wherein the first character of the second
sequence is read. It should be understood that if the sequence is a
nucleotide sequence, then the character would normally be either A,
T, C, G or U. If the sequence is a protein sequence, then it is in
one aspect in the single letter amino acid code so that the first
and sequence sequences can be easily compared.
[0256] A determination is then made at a decision state 264 whether
the two characters are the same. If they are the same, then the
process 250 moves to a state 268 wherein the next characters in the
first and second sequences are read. A determination is then made
whether the next characters are the same. If they are, then the
process 250 continues this loop until two characters are not the
same. If a determination is made that the next two characters are
not the same, the process 250 moves to a decision state 274 to
determine whether there are any more characters either sequence to
read.
[0257] If there are not any more characters to read, then the
process 250 moves to a state 276 wherein the level of homology
between the first and second sequences is displayed to the user.
The level of homology is determined by calculating the proportion
of characters between the sequences that were the same out of the
total number of sequences in the first sequence. Thus, if every
character in a first 100 nucleotide sequence aligned with a every
character in a second sequence, the homology level would be
100%.
[0258] Alternatively, the computer program may be a computer
program which compares the nucleotide sequences of a nucleic acid
sequence as set forth in the invention, to one or more reference
nucleotide sequences in order to determine whether the nucleic acid
code of the invention, differs from a reference nucleic acid
sequence at one or more positions. Optionally such a program
records the length and identity of inserted, deleted or substituted
nucleotides with respect to the sequence of either the reference
polynucleotide or a nucleic acid sequence of the invention. In one
aspect, the computer program may be a program which determines
whether a nucleic acid sequence of the invention, contains a single
nucleotide polymorphism (SNP) with respect to a reference
nucleotide sequence.
[0259] Accordingly, another aspect of the invention is a method for
determining whether a nucleic acid sequence of the invention,
differs at one or more nucleotides from a reference nucleotide
sequence comprising the steps of reading the nucleic acid code and
the reference nucleotide sequence through use of a computer program
which identifies differences between nucleic acid sequences and
identifying differences between the nucleic acid code and the
reference nucleotide sequence with the computer program. In some
aspects, the computer program is a program which identifies single
nucleotide polymorphisms. The method may be implemented by the
computer systems described above and the method illustrated in FIG.
3. The method may also be performed by reading at least 2, 5, 10,
15, 20, 25, 30, or 40 or more of the nucleic acid sequences of the
invention and the reference nucleotide sequences through the use of
the computer program and identifying differences between the
nucleic acid codes and the reference nucleotide sequences with the
computer program.
[0260] In other aspects the computer based system may further
comprise an identifier for identifying features within a nucleic
acid sequence of the invention or a polypeptide sequence of the
invention.
[0261] An "identifier" refers to one or more programs which
identifies certain features within a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention. In one
aspect, the identifier may comprise a program which identifies an
open reading frame in a nucleic acid sequence of the invention.
[0262] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence. The process 300 begins at a start state 302 and then
moves to a state 304 wherein a first sequence that is to be checked
for features is stored to a memory 115 in the computer system 100.
The process 300 then moves to a state 306 wherein a database of
sequence features is opened. Such a database would include a list
of each feature's attributes along with the name of the feature.
For example, a feature name could be "Initiation Codon" and the
attribute would be "ATG". Another example would be the feature name
"TAATAA Box" and the feature attribute would be "TAATAA". An
example of such a database is produced by the University of
Wisconsin Genetics Computer Group. Alternatively, the features may
be structural polypeptide motifs such as alpha helices, beta
sheets, or functional polypeptide motifs such as enzymatic active
sites, helix-turn-helix motifs or other motifs known to those
skilled in the art.
[0263] Once the database of features is opened at the state 306,
the process 300 moves to a state 308 wherein the first feature is
read from the database. A comparison of the attribute of the first
feature with the first sequence is then made at a state 310. A
determination is then made at a decision state 316 whether the
attribute of the feature was found in the first sequence. If the
attribute was found, then the process 300 moves to a state 318
wherein the name of the found feature is displayed to the user.
[0264] The process 300 then moves to a decision state 320 wherein a
determination is made whether move features exist in the database.
If no more features do exist, then the process 300 terminates at an
end state 324. However, if more features do exist in the database,
then the process 300 reads the next sequence feature at a state 326
and loops back to the state 310 wherein the attribute of the next
feature is compared against the first sequence. It should be noted,
that if the feature attribute is not found in the first sequence at
the decision state 316, the process 300 moves directly to the
decision state 320 in order to determine if any more features exist
in the database.
[0265] Accordingly, another aspect of the invention is a method of
identifying a feature within a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention, comprising
reading the nucleic acid code(s) or polypeptide code(s) through the
use of a computer program which identifies features therein and
identifying features within the nucleic acid code(s) with the
computer program. In one aspect, computer program comprises a
computer program which identifies open reading frames. The method
may be performed by reading a single sequence or at least 2, 5, 10,
15, 20, 25, 30, or 40 of the nucleic acid sequences of the
invention, or the polypeptide sequences of the invention, through
the use of the computer program and identifying features within the
nucleic acid codes or polypeptide codes with the computer
program.
[0266] A nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, may be stored and manipulated in a
variety of data processor programs in a variety of formats. For
example, a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, may be stored as text in a word
processing file, such as Microsoft WORD.TM. or WORDPERFECT.TM. or
as an ASCII file in a variety of database programs familiar to
those of skill in the art, such as DB2.TM., SYBASE.TM., or
ORACLE.TM.. In addition, many computer programs and databases may
be used as sequence comparison algorithms, identifiers, or sources
of reference nucleotide sequences or polypeptide sequences to be
compared to a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention. The following list is
intended not to limit the invention but to provide guidance to
programs and databases which are useful with the nucleic acid
sequences of the invention, or the polypeptide sequences of the
invention.
[0267] The programs and databases which may be used include, but
are not limited to: MACPATTERN.TM. (EMBL), DISCOVERYBASE.TM.
(Molecular Applications Group), GENEMINE.TM. (Molecular
Applications Group), LOOK.TM. (Molecular Applications Group),
MACLOOK.TM. (Molecular Applications Group), BLAST and BLAST2
(NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403,
1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:
2444, 1988), FASTDB.TM. (Brutlag et al. Comp. App. Biosci.
6:237-245, 1990), CATALYST.TM. (Molecular Simulations Inc.),
CATALYST.TM./SHAPE.TM. (Molecular Simulations Inc.),
CERIUS.sup.2.DBACCESS.TM. (Molecular Simulations Inc.), HYPOGEN.TM.
(Molecular Simulations Inc.), INSIGHT II.TM., (Molecular
Simulations Inc.), DISCOVER.TM. (Molecular Simulations Inc.),
CHARMm.TM. (Molecular Simulations Inc.), FELIX.TM. (Molecular
Simulations Inc.), DELPHI.TM., (Molecular Simulations Inc.),
QUANTEMM.TM., (Molecular Simulations Inc.), HOMOLOGY.TM. (Molecular
Simulations Inc.), MODELER.TM. (Molecular Simulations Inc.),
ISIS.TM. (Molecular Simulations Inc.), QUANTA.TM./Protein Design
(Molecular Simulations Inc.), WEBLAB.TM. (Molecular Simulations
Inc.), WEBLAB DIVERSITY EXPLORER.TM. (Molecular Simulations Inc.),
GENE EXPLORER.TM. (Molecular Simulations Inc.), SEQFOLD.TM.
(Molecular Simulations Inc.), the MDL Available Chemicals Directory
database, the MDL Drug Data Report data base, the Comprehensive
Medicinal Chemistry database, Derwents' World Drug Index database,
the BioByteMasterFile database, the Genbank database and the
Genseqn database. Many other programs and data bases would be
apparent to one of skill in the art given the present
disclosure.
[0268] Motifs which may be detected using the above programs
include sequences encoding leucine zippers, helix-turn-helix
motifs, glycosylation sites, ubiquitination sites, alpha helices
and beta sheets, signal sequences encoding signal peptides which
direct the secretion of the encoded proteins, sequences implicated
in transcription regulation such as homeoboxes, acidic stretches,
enzymatic active sites, substrate binding sites and enzymatic
cleavage sites.
Hybridization of Nucleic Acids
[0269] The invention provides isolated, synthetic or recombinant
nucleic acids that hybridize under stringent conditions to a
sequence of the invention, including any exemplary sequence of the
invention (e.g., including all odd SEQ ID NO:s between SEQ ID NO:1
and SEQ ID NO:101). The stringent conditions can be highly
stringent conditions, medium stringent conditions and/or low
stringent conditions, including the high and reduced stringency
conditions described herein. In one aspect, it is the stringency of
the wash conditions that set forth the conditions which determine
whether a nucleic acid is within the scope of the invention, as
discussed below.
[0270] "Hybridization" refers to the process by which a nucleic
acid strand joins with a complementary strand through base pairing.
Hybridization reactions can be sensitive and selective so that a
particular sequence of interest can be identified even in samples
in which it is present at low concentrations. Suitably stringent
conditions can be defined by, for example, the concentrations of
salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature and are well known
in the art. In particular, stringency can be increased by reducing
the concentration of salt, increasing the concentration of
formamide, or raising the hybridization temperature. In alternative
aspects, nucleic acids of the invention are defined by their
ability to hybridize under various stringency conditions (e.g.,
high, medium, and low), as set forth herein.
[0271] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In one aspect, hybridization occurs under high
stringency conditions, e.g., at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS and 200 n/ml sheared and denatured salmon
sperm DNA. Hybridization could occur under these reduced stringency
conditions, but in 35% formamide at a reduced temperature of
35.degree. C. The temperature range corresponding to a particular
level of stringency can be further narrowed by calculating the
purine to pyrimidine ratio of the nucleic acid of interest and
adjusting the temperature accordingly. Variations on the above
ranges and conditions are well known in the art.
[0272] In alternative aspects, nucleic acids of the invention as
defined by their ability to hybridize under stringent conditions
can be between about five residues and the full length of nucleic
acid of the invention; e.g., they can be at least 5, 10, 15, 20,
25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, or more, residues in length. Nucleic acids shorter than
full length are also included. These nucleic acids can be useful
as, e.g., hybridization probes, labeling probes, PCR
oligonucleotide probes, iRNA (siRNA or miRNA, single or double
stranded), antisense or sequences encoding antibody binding
peptides (epitopes), motifs, active sites and the like.
[0273] In one aspect, nucleic acids of the invention are defined by
their ability to hybridize under high stringency comprises
conditions of about 50% formamide at about 37.degree. C. to
42.degree. C. In one aspect, nucleic acids of the invention are
defined by their ability to hybridize under reduced stringency
comprising conditions in about 35% to 25% formamide at about
30.degree. C. to 35.degree. C.
[0274] Alternatively, nucleic acids of the invention are defined by
their ability to hybridize under high stringency comprising
conditions at 42.degree. C. in 50% formamide, 5.times.SSPE, 0.3%
SDS, and a repetitive sequence blocking nucleic acid, such as cot-1
or salmon sperm DNA (e.g., 200 .mu.g/ml sheared and denatured
salmon sperm DNA). In one aspect, nucleic acids of the invention
are defined by their ability to hybridize under reduced stringency
conditions comprising 35% formamide at a reduced temperature of
35.degree. C.
[0275] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content) and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0276] Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example
of nucleic acid hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2EDTA, 0.5% SDS,
10.times.Denhardt's and 0.5 mg/ml polyriboadenylic acid.
Approximately 2.times.10.sup.7 cpm (specific activity
4-9.times.10.sup.8 cpm/ug) of .sup.32P end-labeled oligonucleotide
probe are then added to the solution. After 12-16 hours of
incubation, the membrane is washed for 30 minutes at room
temperature in 1.times.SET (150 mM NaCl, 20 mM Tris hydrochloride,
pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed by a 30
minute wash in fresh 1.times.SET at T.sub.m-10.degree. C. for the
oligonucleotide probe. The membrane is then exposed to
auto-radiographic film for detection of hybridization signals.
[0277] All of the foregoing hybridizations would be considered to
be under conditions of high stringency.
[0278] Following hybridization, a filter can be washed to remove
any non-specifically bound detectable probe. The stringency used to
wash the filters can also be varied depending on the nature of the
nucleic acids being hybridized, the length of the nucleic acids
being hybridized, the degree of complementarity, the nucleotide
sequence composition (e.g., GC v. AT content) and the nucleic acid
type (e.g., RNA v. DNA). Examples of progressively higher
stringency condition washes are as follows: 2.times.SSC, 0.1% SDS
at room temperature for 15 minutes (low stringency); 0.1.times.SSC,
0.5% SDS at room temperature for 30 minutes to 1 hour (moderate
stringency); 0.1.times.SSC, 0.5% SDS for 15 to 30 minutes at
between the hybridization temperature and 68.degree. C. (high
stringency); and 0.15M NaCl for 15 minutes at 72.degree. C. (very
high stringency). A final low stringency wash can be conducted in
0.1.times.SSC at room temperature. The examples above are merely
illustrative of one set of conditions that can be used to wash
filters. One of skill in the art would know that there are numerous
recipes for different stringency washes. Some other examples are
given below.
[0279] In one aspect, hybridization conditions comprise a wash step
comprising a wash for 30 minutes at room temperature in a solution
comprising 1.times.150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1
mM Na.sub.2EDTA, 0.5% SDS, followed by a 30 minute wash in fresh
solution.
[0280] Nucleic acids which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0281] The above procedure may be modified to identify nucleic
acids having decreasing levels of homology to the probe sequence.
For example, to obtain nucleic acids of decreasing homology to the
detectable probe, less stringent conditions may be used. For
example, the hybridization temperature may be decreased in
increments of 5.degree. C. from 68.degree. C. to 42.degree. C. in a
hybridization buffer having a Na+ concentration of approximately
1M. Following hybridization, the filter may be washed with
2.times.SSC, 0.5% SDS at the temperature of hybridization. These
conditions are considered to be "moderate" conditions above
50.degree. C. and "low" conditions below 50.degree. C. A specific
example of "moderate" hybridization conditions is when the above
hybridization is conducted at 55.degree. C. A specific example of
"low stringency" hybridization conditions is when the above
hybridization is conducted at 45.degree. C.
[0282] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 10% formamide.
[0283] However, the selection of a hybridization format is not
critical--it is the stringency of the wash conditions that set
forth the conditions which determine whether a nucleic acid is
within the scope of the invention. Wash conditions used to identify
nucleic acids within the scope of the invention include, e.g.: a
salt concentration of about 0.02 molar at pH 7 and a temperature of
at least about 50.degree. C. or about 55.degree. C. to about
60.degree. C.; or, a salt concentration of about 0.15 M NaCl at
72.degree. C. for about 15 minutes; or, a salt concentration of
about 0.2.times.SSC at a temperature of at least about 50.degree.
C. or about 55.degree. C. to about 60.degree. C. for about 15 to
about 20 minutes; or, the hybridization complex is washed twice
with a solution with a salt concentration of about 2.times.SSC
containing 0.1% SDS at room temperature for 15 minutes and then
washed twice by 0.1.times.SSC containing 0.1% SDS at 68.degree. C.
for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen
and Ausubel for a description of SSC buffer and equivalent
conditions.
[0284] These methods may be used to isolate nucleic acids of the
invention. For example, the preceding methods may be used to
isolate nucleic acids having a sequence with at least about 97%, at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 65%, at least 60%, at least 55%, or at least
50% sequence identity (homology) to a nucleic acid sequence
selected from the group consisting of one of the sequences of the
invention, or fragments comprising at least about 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive
bases thereof and the sequences complementary thereto. Sequence
identity (homology) may be measured using the alignment algorithm.
For example, the homologous polynucleotides may have a coding
sequence which is a naturally occurring allelic variant of one of
the coding sequences described herein. Such allelic variants may
have a substitution, deletion or addition of one or more
nucleotides when compared to the nucleic acids of the invention.
Additionally, the above procedures may be used to isolate nucleic
acids which encode polypeptides having at least about 99%, 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 65%, at least 60%, at least 55%, or at least 50% sequence
identity (homology) to a polypeptide of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 consecutive amino acids thereof as determined using a sequence
alignment algorithm (e.g., such as the FASTA version 3.0t78
algorithm with the default parameters).
Oligonucleotides Probes and Methods for Using them
[0285] The invention also provides nucleic acid probes that can be
used, e.g., for identifying nucleic acids encoding a polypeptide
with an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity or
fragments thereof or for identifying ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme genes. In one aspect, the probe
comprises at least 10 consecutive bases of a nucleic acid of the
invention. Alternatively, a probe of the invention can be at least
about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110,
120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70,
consecutive bases of a sequence as set forth in a nucleic acid of
the invention. The probes identify a nucleic acid by binding and/or
hybridization. The probes can be used in arrays of the invention,
see discussion below, including, e.g., capillary arrays. The probes
of the invention can also be used to isolate other nucleic acids or
polypeptides.
[0286] The isolated nucleic acids of the invention, the sequences
complementary thereto, or a fragment comprising at least 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of one of the sequences of the invention, or the
sequences complementary thereto may also be used as probes to
determine whether a biological sample, such as a soil sample,
contains an organism having a nucleic acid sequence of the
invention or an organism from which the nucleic acid was obtained.
In such procedures, a biological sample potentially harboring the
organism from which the nucleic acid was isolated is obtained and
nucleic acids are obtained from the sample. The nucleic acids are
contacted with the probe under conditions which permit the probe to
specifically hybridize to any complementary sequences from which
are present therein.
[0287] Where necessary, conditions which permit the probe to
specifically hybridize to complementary sequences may be determined
by placing the probe in contact with complementary sequences from
samples known to contain the complementary sequence as well as
control sequences which do not contain the complementary sequence.
Hybridization conditions, such as the salt concentration of the
hybridization buffer, the formamide concentration of the
hybridization buffer, or the hybridization temperature, may be
varied to identify conditions which allow the probe to hybridize
specifically to complementary nucleic acids.
[0288] If the sample contains the organism from which the nucleic
acid was isolated, specific hybridization of the probe is then
detected. Hybridization may be detected by labeling the probe with
a detectable agent such as a radioactive isotope, a fluorescent dye
or an enzyme capable of catalyzing the formation of a detectable
product.
[0289] Many methods for using the labeled probes to detect the
presence of complementary nucleic acids in a sample are familiar to
those skilled in the art. These include Southern Blots, Northern
Blots, colony hybridization procedures and dot blots. Protocols for
each of these procedures are provided in Ausubel et al. Current
Protocols in Molecular Biology, John Wiley 503 Sons, Inc. (1997)
and Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd
Ed., Cold Spring Harbor Laboratory Press (1989.
[0290] Alternatively, more than one probe (at least one of which is
capable of specifically hybridizing to any complementary sequences
which are present in the nucleic acid sample), may be used in an
amplification reaction to determine whether the sample contains an
organism containing a nucleic acid sequence of the invention (e.g.,
an organism from which the nucleic acid was isolated). Typically,
the probes comprise oligonucleotides. In one aspect, the
amplification reaction may comprise a PCR reaction. PCR protocols
are described in Ausubel and Sambrook, supra. Alternatively, the
amplification may comprise a ligase chain reaction, 3SR, or strand
displacement reaction. (See Barany, F., "The Ligase Chain Reaction
in a PCR World", PCR Methods and Applications 1:5-16, 1991; E. Fahy
et al., "Self-sustained Sequence Replication (3SR): An Isothermal
Transcription-based Amplification System Alternative to PCR", PCR
Methods and Applications 1:25-33, 1991; and Walker G. T. et al.,
"Strand Displacement Amplification--an Isothermal in vitro DNA
Amplification Technique", Nucleic Acid Research 20:1691-1696,
1992). In such procedures, the nucleic acids in the sample are
contacted with the probes, the amplification reaction is performed
and any resulting amplification product is detected. The
amplification product may be detected by performing gel
electrophoresis on the reaction products and staining the gel with
an intercalator such as ethidium bromide. Alternatively, one or
more of the probes may be labeled with a radioactive isotope and
the presence of a radioactive amplification product may be detected
by autoradiography after gel electrophoresis.
[0291] Probes derived from sequences near the ends of the sequences
of the invention, may also be used in chromosome walking procedures
to identify clones containing genomic sequences located adjacent to
the sequences of the invention. Such methods allow the isolation of
genes which encode additional proteins from the host organism.
[0292] The isolated nucleic acids of the invention, the sequences
complementary thereto, or a fragment comprising at least 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of one of the sequences of the invention, or the
sequences complementary thereto may be used as probes to identify
and isolate related nucleic acids. In some aspects, the related
nucleic acids may be cDNAs or genomic DNAs from organisms other
than the one from which the nucleic acid was isolated. For example,
the other organisms may be related organisms. In such procedures, a
nucleic acid sample is contacted with the probe under conditions
which permit the probe to specifically hybridize to related
sequences. Hybridization of the probe to nucleic acids from the
related organism is then detected using any of the methods
described above.
[0293] By varying the stringency of the hybridization conditions
used to identify nucleic acids, such as cDNAs or genomic DNAs,
which hybridize to the detectable probe, nucleic acids having
different levels of homology to the probe can be identified and
isolated. Stringency may be varied by conducting the hybridization
at varying temperatures below the melting temperatures of the
probes. The melting temperature, T.sub.m, is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly complementary probe. Very stringent
conditions are selected to be equal to or about 5.degree. C. lower
than the T.sub.m for a particular probe. The melting temperature of
the probe may be calculated using the following formulas:
[0294] For probes between 14 and 70 nucleotides in length the
melting temperature (T.sub.m) is calculated using the formula:
T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(600/N) where N is
the length of the probe.
[0295] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation: T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(0.63%
formamide)-(600/N) where N is the length of the probe.
[0296] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 100 .mu.g/ml denatured fragmented salmon sperm
DNA, 50% formamide. The formulas for SSC and Denhardt's solutions
are listed in Sambrook et al., supra.
[0297] Hybridization is conducted by adding the detectable probe to
the prehybridization solutions listed above. Where the probe
comprises double stranded DNA, it is denatured before addition to
the hybridization solution. The filter is contacted with the
hybridization solution for a sufficient period of time to allow the
probe to hybridize to cDNAs or genomic DNAs containing sequences
complementary thereto or homologous thereto. For probes over 200
nucleotides in length, the hybridization may be carried out at
15-25.degree. C. below the T.sub.m. For shorter probes, such as
oligonucleotide probes, the hybridization may be conducted at
5-10.degree. C. below the T.sub.m. In one aspect, for
hybridizations in 6.times.SSC, the hybridization is conducted at
approximately 68.degree. C. Usually, for hybridizations in 50%
formamide containing solutions, the hybridization is conducted at
approximately 42.degree. C.
Inhibiting Expression of Ammonia Lyase, e.g., Phenylalanine Ammonia
Lyase, Tyrosine Ammonia Lyase and/or Histidine Ammonia Lyase
Enzymes
[0298] The invention provides nucleic acids complementary to (e.g.,
antisense sequences to) the nucleic acids of the invention, e.g.,
ammonia lyase enzyme-encoding nucleic acids, e.g., nucleic acids
comprising antisense, iRNA, ribozymes. Nucleic acids of the
invention comprising antisense sequences can be capable of
inhibiting the transport, splicing or transcription of ammonia
lyase enzyme-encoding genes. The inhibition can be effected through
the targeting of genomic DNA or messenger RNA. The transcription or
function of targeted nucleic acid can be inhibited, for example, by
hybridization and/or cleavage. One particularly useful set of
inhibitors provided by the present invention includes
oligonucleotides which are able to either bind ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme gene or message, in either case
preventing or inhibiting the production or function of an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme. The association can be
through sequence specific hybridization. Another useful class of
inhibitors includes oligonucleotides which cause inactivation or
cleavage of ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
message. The oligonucleotide can have enzyme activity which causes
such cleavage, such as ribozymes. The oligonucleotide can be
chemically modified or conjugated to an enzyme or composition
capable of cleaving the complementary nucleic acid. A pool of many
different such oligonucleotides can be screened for those with the
desired activity. Thus, the invention provides various compositions
for the inhibition of ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
expression on a nucleic acid and/or protein level, e.g., antisense,
iRNA (e.g., siRNA, miRNA) and ribozymes comprising ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme sequences of the invention and the
anti-ammonia lyase, e.g., anti-phenylalanine ammonia lyase,
anti-tyrosine ammonia lyase and/or anti-histidine ammonia lyase
enzyme antibodies of the invention.
[0299] Inhibition of ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
expression can have a variety of industrial applications. For
example, inhibition of ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
expression can slow or prevent spoilage. In one aspect, use of
compositions of the invention that inhibit the expression and/or
activity of ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes,
e.g., antibodies, antisense oligonucleotides, ribozymes and RNAi,
are used to slow or prevent spoilage. Thus, in one aspect, the
invention provides methods and compositions comprising application
onto a plant or plant product (e.g., a cereal, a grain, a fruit,
seed, root, leaf, etc.) antibodies, antisense oligonucleotides,
ribozymes and RNAi of the invention to slow or prevent spoilage.
These compositions also can be expressed by the plant (e.g., a
transgenic plant) or another organism (e.g., a bacterium or other
microorganism transformed with an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme gene of the invention).
[0300] The compositions of the invention for the inhibition of
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme expression (e.g.,
antisense, iRNA, ribozymes, antibodies) can be used as
pharmaceutical compositions, e.g., as anti-pathogen agents or in
other therapies, e.g., as anti-microbials for, e.g.,
Salmonella.
[0301] Antisense Oligonucleotides
[0302] The invention provides antisense oligonucleotides capable of
binding ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme message which,
in one aspect, can inhibit ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity by targeting mRNA. Strategies for designing
antisense oligonucleotides are well described in the scientific and
patent literature, and the skilled artisan can design such ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme oligonucleotides using the
novel reagents of the invention. For example, gene walking/RNA
mapping protocols to screen for effective antisense
oligonucleotides are well known in the art, see, e.g., Ho (2000)
Methods Enzymol. 314:168-183, describing an RNA mapping assay,
which is based on standard molecular techniques to provide an easy
and reliable method for potent antisense sequence selection. See
also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.
[0303] Naturally occurring nucleic acids are used as antisense
oligonucleotides. The antisense oligonucleotides can be of any
length; for example, in alternative aspects, the antisense
oligonucleotides are between about 5 to 100, about 10 to 80, about
15 to 60, about 18 to 40. The optimal length can be determined by
routine screening. The antisense oligonucleotides can be present at
any concentration. The optimal concentration can be determined by
routine screening. A wide variety of synthetic, non-naturally
occurring nucleotide and nucleic acid analogues are known which can
address this potential problem. For example, peptide nucleic acids
(PNAs) containing non-ionic backbones, such as N-(2-aminoethyl)
glycine units can be used. Antisense oligonucleotides having
phosphorothioate linkages can also be used, as described in WO
97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol
144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press,
Totowa, N.J., 1996). Antisense oligonucleotides having synthetic
DNA backbone analogues provided by the invention can also include
phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl
phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino),
3'-N-carbamate, and morpholino carbamate nucleic acids, as
described above.
[0304] Combinatorial chemistry methodology can be used to create
vast numbers of oligonucleotides that can be rapidly screened for
specific oligonucleotides that have appropriate binding affinities
and specificities toward any target, such as the sense and
antisense ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
sequences of the invention (see, e.g., Gold (1995) J. of Biol.
Chem. 270:13581-13584).
[0305] Inhibitory Ribozymes
[0306] The invention provides ribozymes capable of binding ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme message. These ribozymes can
inhibit ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity by,
e.g., targeting mRNA. Strategies for designing ribozymes and
selecting the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase
enzyme-specific antisense sequence for targeting are well described
in the scientific and patent literature, and the skilled artisan
can design such ribozymes using the novel reagents of the
invention. Ribozymes act by binding to a target RNA through the
target RNA binding portion of a ribozyme which is held in close
proximity to an enzymatic portion of the RNA that cleaves the
target RNA. Thus, the ribozyme recognizes and binds a target RNA
through complementary base-pairing, and once bound to the correct
site, acts enzymatically to cleave and inactivate the target RNA.
Cleavage of a target RNA in such a manner will destroy its ability
to direct synthesis of an encoded protein if the cleavage occurs in
the coding sequence. After a ribozyme has bound and cleaved its RNA
target, it can be released from that RNA to bind and cleave new
targets repeatedly.
[0307] In some circumstances, the enzymatic nature of a ribozyme
can be advantageous over other technologies, such as antisense
technology (where a nucleic acid molecule simply binds to a nucleic
acid target to block its transcription, translation or association
with another molecule) as the effective concentration of ribozyme
necessary to effect a therapeutic treatment can be lower than that
of an antisense oligonucleotide. This potential advantage reflects
the ability of the ribozyme to act enzymatically. Thus, a single
ribozyme molecule is able to cleave many molecules of target RNA.
In addition, a ribozyme is typically a highly specific inhibitor,
with the specificity of inhibition depending not only on the base
pairing mechanism of binding, but also on the mechanism by which
the molecule inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA target and
so specificity is defined as the ratio of the rate of cleavage of
the targeted RNA over the rate of cleavage of non-targeted RNA.
This cleavage mechanism is dependent upon factors additional to
those involved in base pairing. Thus, the specificity of action of
a ribozyme can be greater than that of antisense oligonucleotide
binding the same RNA site.
[0308] The ribozyme of the invention, e.g., an enzymatic ribozyme
RNA molecule, can be formed in a hammerhead motif, a hairpin motif,
as a hepatitis delta virus motif, a group I intron motif and/or an
RNaseP-like RNA in association with an RNA guide sequence. Examples
of hammerhead motifs are described by, e.g., Rossi (1992) Aids
Research and Human Retroviruses 8:183; hairpin motifs by Hampel
(1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
18:299; the hepatitis delta virus motif by Perrotta (1992)
Biochemistry 31:16; the RNaseP motif by Guerrier-Takada (1983) Cell
35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The
recitation of these specific motifs is not intended to be limiting.
Those skilled in the art will recognize that a ribozyme of the
invention, e.g., an enzymatic RNA molecule of this invention, can
have a specific substrate binding site complementary to one or more
of the target gene RNA regions. A ribozyme of the invention can
have a nucleotide sequence within or surrounding that substrate
binding site which imparts an RNA cleaving activity to the
molecule.
[0309] RNA Interference (RNAi)
[0310] In one aspect, the invention provides an RNA inhibitory
molecule, a so-called "RNAi" molecule, comprising an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme sequence of the invention. The RNAi
molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi
molecule, e.g., siRNA and/or miRNA, can inhibit expression of an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme gene. In one aspect,
the RNAi molecule, e.g., siRNA and/or miRNA, is about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
[0311] While the invention is not limited by any particular
mechanism of action, the RNAi can enter a cell and cause the
degradation of a single-stranded RNA (ssRNA) of similar or
identical sequences, including endogenous mRNAs. When a cell is
exposed to double-stranded RNA (dsRNA), mRNA from the homologous
gene is selectively degraded by a process called RNA interference
(RNAi). A possible basic mechanism behind RNAi is the breaking of a
double-stranded RNA (dsRNA) matching a specific gene sequence into
short pieces called short interfering RNA, which trigger the
degradation of mRNA that matches its sequence. In one aspect, the
RNAi's of the invention are used in gene-silencing therapeutics,
see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one
aspect, the invention provides methods to selectively degrade RNA
using the RNAi's molecules, e.g., siRNA and/or miRNA, of the
invention. In one aspect, the micro-inhibitory RNA (miRNA) inhibits
translation, and the siRNA inhibits transcription. The process may
be practiced in vitro, ex vivo or in vivo. In one aspect, the RNAi
molecules of the invention can be used to generate a
loss-of-function mutation in a cell, an organ or an animal. Methods
for making and using RNAi molecules, e.g., siRNA and/or miRNA, for
selectively degrade RNA are well known in the art, see, e.g., U.S.
Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
Modification of Nucleic Acids
[0312] The invention provides methods of generating variants of the
nucleic acids of the invention, e.g., those encoding an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme. These methods can be
repeated or used in various combinations to generate ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes having an altered or different
activity or an altered or different stability from that of an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme encoded by the template
nucleic acid. These methods also can be repeated or used in various
combinations, e.g., to generate variations in gene/message
expression, message translation or message stability. In another
aspect, the genetic composition of a cell is altered by, e.g.,
modification of a homologous gene ex vivo, followed by its
reinsertion into the cell.
[0313] A nucleic acid of the invention can be altered by any means.
For example, random or stochastic methods, or, non-stochastic, or
"directed evolution," methods, see, e.g., U.S. Pat. No. 6,361,974.
Methods for random mutation of genes are well known in the art,
see, e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be
used to randomly mutate a gene. Mutagens include, e.g., ultraviolet
light or gamma irradiation, or a chemical mutagen, e.g., mitomycin,
nitrous acid, photoactivated psoralens, alone or in combination, to
induce DNA breaks amenable to repair by recombination. Other
chemical mutagens include, for example, sodium bisulfite, nitrous
acid, hydroxylamine, hydrazine or formic acid. Other mutagens are
analogues of nucleotide precursors, e.g., nitrosoguanidine,
5-bromouracil, 2-aminopurine, or acridine. These agents can be
added to a PCR reaction in place of the nucleotide precursor
thereby mutating the sequence. Intercalating agents such as
proflavine, acriflavine, quinacrine and the like can also be
used.
[0314] Any technique in molecular biology can be used, e.g., random
PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA
89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,
e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively,
nucleic acids, e.g., genes, can be reassembled after random, or
"stochastic," fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242;
6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238;
5,605,793. In alternative aspects, modifications, additions or
deletions are introduced by error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR),
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, and/or a combination of
these and other methods.
[0315] In one aspect, a metagenomic discovery and a non-stochastic
method of directed evolution (called "DIRECTEVOLUTION.RTM., as
described, e.g., in U.S. Pat. No. 6,939,689, which includes Gene
Site Saturation Mutagenesis (GSSM) (as discussed above, see also
U.S. Pat. Nos. 6,171,820 and 6,579,258) and Tunable GeneReassembly
(TGR) (see, e.g., U.S. Pat. No. 6,537,776) technology is used to
practice the invention, e.g., for the discovery and/or optimization
of lyases of the invention.
[0316] The following publications describe a variety of recursive
recombination procedures and/or methods which can be incorporated
into the methods of the invention: Stemmer (1999) "Molecular
breeding of viruses for targeting and other clinical properties"
Tumor Targeting 4:1-4; Ness (1999) Nature Biotechnology 17:893-896;
Chang (1999) "Evolution of a cytokine using DNA family shuffling"
Nature Biotechnology 17:793-797; Minshull (1999) "Protein evolution
by molecular breeding" Current Opinion in Chemical Biology
3:284-290; Christians (1999) "Directed evolution of thymidine
kinase for AZT phosphorylation using DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri (1998) "DNA shuffling of a family
of genes from diverse species accelerates directed evolution"
Nature 391:288-291; Crameri (1997) "Molecular evolution of an
arsenate detoxification pathway by DNA shuffling," Nature
Biotechnology 15:436-438; Zhang (1997) "Directed evolution of an
effective fucosidase from a galactosidase by DNA shuffling and
screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al.
(1997) "Applications of DNA Shuffling to Pharmaceuticals and
Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et
al. (1996) "Construction and evolution of antibody-phage libraries
by DNA shuffling" Nature Medicine 2:100-103; Gates et al. (1996)
"Affinity selective isolation of ligands from peptide libraries
through display on a lac repressor `headpiece dimer`" Journal of
Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and
Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH
Publishers, New York. pp. 447-457; Crameri and Stemmer (1995)
"Combinatorial multiple cassette mutagenesis creates all the
permutations of mutant and wildtype cassettes" BioTechniques
18:194-195; Stemmer et al. (1995) "Single-step assembly of a gene
and entire plasmid form large numbers of oligodeoxyribonucleotides"
Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular
Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence
Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution
of a protein in vitro by DNA shuffling" Nature 370:389-391; and
Stemmer (1994) "DNA shuffling by random fragmentation and
reassembly: In vitro recombination for molecular evolution." Proc.
Natl. Acad. Sci. USA 91:10747-10751.
[0317] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al. (1997) "Approaches
to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178;
Dale et al. (1996) "Oligonucleotide-directed random mutagenesis
using the phosphorothioate method" Methods Mol. Biol. 57:369-374;
Smith (1985) "In vitro mutagenesis" Ann Rev. Genet. 19:423-462;
Botstein & Shortle (1985) "Strategies and applications of in
vitro mutagenesis" Science 229:1193-1201; Carter (1986)
"Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987)
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)); mutagenesis using uracil
containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and
efficient site-specific mutagenesis without phenotypic selection"
Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp repressors with new DNA-binding specificities" Science
242:240-245); oligonucleotide-directed mutagenesis (Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350
(1987); Zoller (1982) "Oligonucleotide-directed mutagenesis using
M13-derived vectors: an efficient and general procedure for the
production of point mutations in any DNA fragment" Nucleic Acids
Res. 10:6487-6500; Zoller & Smith (1983)
"Oligonucleotide-directed mutagenesis of DNA fragments cloned into
M13 vectors" Methods in Enzymol. 100:468-500; and Zoller (1987)
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template" Methods
in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis
(Taylor (1985) "The use of phosphorothioate-modified DNA in
restriction enzyme reactions to prepare nicked DNA" Nucl. Acids
Res. 13: 8749-8764; Taylor (1985) "The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye (1986) "Inhibition of restriction endonuclease Nci
I cleavage by phosphorothioate groups and its application to
oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14:
9679-9698; Sayers (1988) "Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl.
Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide"
Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA
(Kramer et al. (1984) "The gapped duplex DNA approach to
oligonucleotide-directed mutation construction" Nucl. Acids Res.
12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.
"Oligonucleotide-directed construction of mutations via gapped
duplex DNA" 154:350-367; Kramer (1988) "Improved enzymatic in vitro
reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids
Res. 16: 7207; and Fritz (1988) "Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
[0318] Additional protocols that can be used to practice the
invention include point mismatch repair (Kramer (1984) "Point
Mismatch Repair" Cell 38:879-887), mutagenesis using
repair-deficient host strains (Carter et al. (1985) "Improved
oligonucleotide site-directed mutagenesis using M13 vectors" Nucl.
Acids Res. 13: 4431-4443; and Carter (1987) "Improved
oligonucleotide-directed mutagenesis using M13 vectors" Methods in
Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986)
"Use of oligonucleotides to generate large deletions" Nucl. Acids
Res. 14: 5115), restriction-selection and restriction-selection and
restriction-purification (Wells et al. (1986) "Importance of
hydrogen-bond formation in stabilizing the transition state of
subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis
by total gene synthesis (Nambiar et al. (1984) "Total synthesis and
cloning of a gene coding for the ribonuclease S protein" Science
223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and
expression of a gene for the a-subunit of bovine rod outer segment
guanine nucleotide-binding protein (transducin)" Nucl. Acids Res.
14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an
efficient method for generation of multiple mutations at defined
sites" Gene 34:315-323; and Grundstrom et al. (1985)
"Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break
repair (Mandecki (1986); Arnold (1993) "Protein engineering for
unusual environments" Current Opinion in Biotechnology 4:450-455.
"Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis" Proc.
Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of
the above methods can be found in Methods in Enzymology Volume 154,
which also describes useful controls for trouble-shooting problems
with various mutagenesis methods.
[0319] Protocols that can be used to practice the invention are
described, e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,
1997), "Methods for In Vitro Recombination;" U.S. Pat. No.
5,811,238 to Stemmer et al. (Sep. 22, 1998) "Methods for Generating
Polynucleotides having Desired Characteristics by Iterative
Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et
al. (Nov. 3, 1998), "DNA Mutagenesis by Random Fragmentation and
Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10,
1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No.
5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and
Compositions for Cellular and Metabolic Engineering;" WO 95/22625,
Stemmer and Crameri, "Mutagenesis by Random Fragmentation and
Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End
Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer
and Crameri "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" WO
97/35966 by Minshull and Stemmer, "Methods and Compositions for
Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al.
"Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et
al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al.
"Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et
al. "Optimization of Immunomodulatory Properties of Genetic
Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by
Random Fragmentation and Reassembly;" EP 0932670 by Stemmer
"Evolving Cellular DNA Uptake by Recursive Sequence Recombination;"
WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and
Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al.,
"Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al.
"Evolution of Whole Cells and Organisms by Recursive Sequence
Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and
Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer
et al., "Methods for Optimization of Gene Therapy by Recursive
Sequence Shuffling and Selection," WO 00/00632, "Methods for
Generating Highly Diverse Libraries," WO 00/09679, "Methods for
Obtaining in Vitro Recombined Polynucleotide Sequence Banks and
Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination
of Polynucleotide Sequences Using Random or Defined Primers," WO
99/29902 by Arnold et al., "Method for Creating Polynucleotide and
Polypeptide Sequences," WO 98/41653 by Vind, "An in Vitro Method
for Construction of a DNA Library," WO 98/41622 by Borchert et al.,
"Method for Constructing a Library Using DNA Shuffling," and WO
98/42727 by Pati and Zarling, "Sequence Alterations using
Homologous Recombination."
[0320] Protocols that can be used to practice the invention
(providing details regarding various diversity generating methods)
are described, e.g., in U.S. patent application Ser. No.
09/407,800, "SHUFFLING OF CODON ALTERED GENES" by Patten et al.
filed Sep. 28, 1999; "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY
RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre et al., U.S. Pat.
No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" by Crameri et al., U.S. Pat. Nos. 6,319,714;
6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; "USE
OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING"
by Welch et al., U.S. Pat. No. 6,436,675; "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING
DESIRED CHARACTERISTICS" by Selifonov et al., filed Jan. 18, 2000,
(PCT/US00/01202) and, e.g. "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS"
by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.
09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN
EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed Jan. 18,
2000 (PCT/US00/01138); and "SINGLE-STRANDED NUCLEIC ACID
TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT
ISOLATION" by Affholter, filed Sep. 6, 2000 (U.S. Ser. No.
09/656,549); and U.S. Pat. Nos. 6,177,263; 6,153,410.
[0321] Non-stochastic, or "directed evolution," methods include,
e.g., saturation mutagenesis, such as Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a
combination thereof are used to modify the nucleic acids of the
invention to generate ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzymes with new or altered properties (e.g., activity under highly
acidic or alkaline conditions, high or low temperatures, and the
like). Polypeptides encoded by the modified nucleic acids can be
screened for an activity before testing for glucan hydrolysis or
other activity. Any testing modality or protocol can be used, e.g.,
using a capillary array platform. See, e.g., U.S. Pat. Nos.
6,361,974; 6,280,926; 5,939,250.
[0322] Gene Site Saturation Mutagenesis, or, GSSM
[0323] The invention also provides methods for making enzyme using
Gene Site Saturation mutagenesis, or, GSSM, as described herein,
and also in U.S. Pat. Nos. 6,171,820 and 6,579,258.
[0324] In one aspect, codon primers containing a degenerate N,N,G/T
sequence are used to introduce point mutations into a
polynucleotide, e.g., an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
or an antibody of the invention, so as to generate a set of progeny
polypeptides in which a full range of single amino acid
substitutions is represented at each amino acid position, e.g., an
amino acid residue in an enzyme active site or ligand binding site
targeted to be modified. These oligonucleotides can comprise a
contiguous first homologous sequence, a degenerate N,N,G/T
sequence, and, optionally, a second homologous sequence. The
downstream progeny translational products from the use of such
oligonucleotides include all possible amino acid changes at each
amino acid site along the polypeptide, because the degeneracy of
the N,N,G/T sequence includes codons for all 20 amino acids. In one
aspect, one such degenerate oligonucleotide (comprised of, e.g.,
one degenerate N,N,G/T cassette) is used for subjecting each
original codon in a parental polynucleotide template to a full
range of codon substitutions. In another aspect, at least two
degenerate cassettes are used--either in the same oligonucleotide
or not, for subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions. For
example, more than one N,N,G/T sequence can be contained in one
oligonucleotide to introduce amino acid mutations at more than one
site. This plurality of N,N,G/T sequences can be directly
contiguous, or separated by one or more additional nucleotide
sequence(s). In another aspect, oligonucleotides serviceable for
introducing additions and deletions can be used either alone or in
combination with the codons containing an N,N,G/T sequence, to
introduce any combination or permutation of amino acid additions,
deletions, and/or substitutions.
[0325] In one aspect, simultaneous mutagenesis of two or more
contiguous amino acid positions is done using an oligonucleotide
that contains contiguous N,N,G/T triplets, i.e. a degenerate
(N,N,G/T)n sequence. In another aspect, degenerate cassettes having
less degeneracy than the N,N,G/T sequence are used. For example, it
may be desirable in some instances to use (e.g. in an
oligonucleotide) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
[0326] In one aspect, use of degenerate triplets (e.g., N,N,G/T
triplets) allows for systematic and easy generation of a full range
of possible natural amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide (in
alternative aspects, the methods also include generation of less
than all possible substitutions per amino acid residue, or codon,
position). For example, for a 100 amino acid polypeptide, 2000
distinct species (i.e. 20 possible amino acids per
position.times.100 amino acid positions) can be generated. Through
the use of an oligonucleotide or set of oligonucleotides containing
a degenerate N,N,G/T triplet, 32 individual sequences can code for
all 20 possible natural amino acids. Thus, in a reaction vessel in
which a parental polynucleotide sequence is subjected to saturation
mutagenesis using at least one such oligonucleotide, there are
generated 32 distinct progeny polynucleotides encoding 20 distinct
polypeptides. In contrast, the use of a non-degenerate
oligonucleotide in site-directed mutagenesis leads to only one
progeny polypeptide product per reaction vessel. Nondegenerate
oligonucleotides can optionally be used in combination with
degenerate primers disclosed; for example, nondegenerate
oligonucleotides can be used to generate specific point mutations
in a working polynucleotide. This provides one means to generate
specific silent point mutations, point mutations leading to
corresponding amino acid changes, and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0327] In one aspect, each saturation mutagenesis reaction vessel
contains polynucleotides encoding at least 20 progeny polypeptide
(e.g., ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes) molecules
such that all 20 natural amino acids are represented at the one
specific amino acid position corresponding to the codon position
mutagenized in the parental polynucleotide (other aspects use less
than all 20 natural combinations). The 32-fold degenerate progeny
polypeptides generated from each saturation mutagenesis reaction
vessel can be subjected to clonal amplification (e.g. cloned into a
suitable host, e.g., E. coli host, using, e.g., an expression
vector) and subjected to expression screening. When an individual
progeny polypeptide is identified by screening to display a
favorable change in property (when compared to the parental
polypeptide, such as increased glucan hydrolysis activity under
alkaline or acidic conditions), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[0328] In one aspect, upon mutagenizing each and every amino acid
position in a parental polypeptide using saturation mutagenesis as
disclosed herein, favorable amino acid changes may be identified at
more than one amino acid position. One or more new progeny
molecules can be generated that contain a combination of all or
part of these favorable amino acid substitutions. For example, if 2
specific favorable amino acid changes are identified in each of 3
amino acid positions in a polypeptide, the permutations include 3
possibilities at each position (no change from the original amino
acid, and each of two favorable changes) and 3 positions. Thus,
there are 3.times.3.times.3 or 27 total possibilities, including 7
that were previously examined--6 single point mutations (i.e. 2 at
each of three positions) and no change at any position.
[0329] In yet another aspect, site-saturation mutagenesis can be
used together with shuffling, chimerization, recombination and
other mutagenizing processes, along with screening. This invention
provides for the use of any mutagenizing process(es), including
saturation mutagenesis, in an iterative manner. In one
exemplification, the iterative use of any mutagenizing process(es)
is used in combination with screening.
[0330] The invention also provides for the use of proprietary codon
primers (containing a degenerate N,N,N sequence) to introduce point
mutations into a polynucleotide, so as to generate a set of progeny
polypeptides in which a full range of single amino acid
substitutions is represented at each amino acid position (Gene Site
Saturation Mutagenesis (GSSM)). The oligos used are comprised
contiguously of a first homologous sequence, a degenerate N,N,N
sequence and in one aspect but not necessarily a second homologous
sequence. The downstream progeny translational products from the
use of such oligos include all possible amino acid changes at each
amino acid site along the polypeptide, because the degeneracy of
the N,N,N sequence includes codons for all 20 amino acids.
[0331] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,N cassette) is used for subjecting each original
codon in a parental polynucleotide template to a full range of
codon substitutions. In another aspect, at least two degenerate
N,N,N cassettes are used--either in the same oligo or not, for
subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions.
Thus, more than one N,N,N sequence can be contained in one oligo to
introduce amino acid mutations at more than one site. This
plurality of N,N,N sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligos serviceable for introducing additions and
deletions can be used either alone or in combination with the
codons containing an N,N,N sequence, to introduce any combination
or permutation of amino acid additions, deletions and/or
substitutions.
[0332] In a particular exemplification, it is possible to
simultaneously mutagenize two or more contiguous amino acid
positions using an oligo that contains contiguous N,N,N triplets,
i.e. a degenerate (N,N,N).sub.n sequence.
[0333] In another aspect, the present invention provides for the
use of degenerate cassettes having less degeneracy than the N,N,N
sequence. For example, it may be desirable in some instances to use
(e.g. in an oligo) a degenerate triplet sequence comprised of only
one N, where the N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g., in an oligo) a degenerate N,N,N triplet sequence,
N,N,G/T, or an N,N, G/C triplet sequence.
[0334] It is appreciated, however, that the use of a degenerate
triplet (such as N,N,G/T or an N,N, G/C triplet sequence) as
disclosed in the instant invention is advantageous for several
reasons. In one aspect, this invention provides a means to
systematically and fairly easily generate the substitution of the
full range of possible amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide. Thus, for
a 100 amino acid polypeptide, the invention provides a way to
systematically and fairly easily generate 2000 distinct species
(i.e., 20 possible amino acids per position times 100 amino acid
positions). It is appreciated that there is provided, through the
use of an oligo containing a degenerate N,N,G/T or an N,N, G/C
triplet sequence, 32 individual sequences that code for 20 possible
amino acids. Thus, in a reaction vessel in which a parental
polynucleotide sequence is subjected to saturation mutagenesis
using one such oligo, there are generated 32 distinct progeny
polynucleotides encoding 20 distinct polypeptides. In contrast, the
use of a non-degenerate oligo in site-directed mutagenesis leads to
only one progeny polypeptide product per reaction vessel.
[0335] This invention also provides for the use of nondegenerate
oligos, which can optionally be used in combination with degenerate
primers disclosed. It is appreciated that in some situations, it is
advantageous to use nondegenerate oligos to generate specific point
mutations in a working polynucleotide. This provides a means to
generate specific silent point mutations, point mutations leading
to corresponding amino acid changes and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0336] Thus, in one aspect of this invention, each saturation
mutagenesis reaction vessel contains polynucleotides encoding at
least 20 progeny polypeptide molecules such that all 20 amino acids
are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide. The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g., cloned into a suitable E.
coli host using an expression vector) and subjected to expression
screening. When an individual progeny polypeptide is identified by
screening to display a favorable change in property (when compared
to the parental polypeptide), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[0337] It is appreciated that upon mutagenizing each and every
amino acid position in a parental polypeptide using saturation
mutagenesis as disclosed herein, favorable amino acid changes may
be identified at more than one amino acid position. One or more new
progeny molecules can be generated that contain a combination of
all or part of these favorable amino acid substitutions. For
example, if 2 specific favorable amino acid changes are identified
in each of 3 amino acid positions in a polypeptide, the
permutations include 3 possibilities at each position (no change
from the original amino acid and each of two favorable changes) and
3 positions. Thus, there are 3.times.3.times.3 or 27 total
possibilities, including 7 that were previously examined--6 single
point mutations (i.e., 2 at each of three positions) and no change
at any position.
[0338] Thus, in a non-limiting exemplification, this invention
provides for the use of saturation mutagenesis in combination with
additional mutagenization processes, such as process where two or
more related polynucleotides are introduced into a suitable host
cell such that a hybrid polynucleotide is generated by
recombination and reductive reassortment.
[0339] In addition to performing mutagenesis along the entire
sequence of a gene, the instant invention provides that mutagenesis
can be use to replace each of any number of bases in a
polynucleotide sequence, wherein the number of bases to be
mutagenized is in one aspect every integer from 15 to 100,000.
Thus, instead of mutagenizing every position along a molecule, one
can subject every or a discrete number of bases (in one aspect a
subset totaling from 15 to 100,000) to mutagenesis. In one aspect,
a separate nucleotide is used for mutagenizing each position or
group of positions along a polynucleotide sequence. A group of 3
positions to be mutagenized may be a codon. The mutations can be
introduced using a mutagenic primer, containing a heterologous
cassette, also referred to as a mutagenic cassette. Exemplary
cassettes can have from 1 to 500 bases. Each nucleotide position in
such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G,
C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base
that is not A, C, G, or T (E can be referred to as a designer
oligo).
[0340] In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is in one aspect about 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is in one aspect from about 15 to 100,000 bases in
length). Thus, a group of mutations (ranging from 1 to 100
mutations) is introduced into each cassette to be mutagenized. A
grouping of mutations to be introduced into one cassette can be
different or the same from a second grouping of mutations to be
introduced into a second cassette during the application of one
round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons and groupings
of particular nucleotide cassettes.
[0341] Defined sequences to be mutagenized include a whole gene,
pathway, cDNA, an entire open reading frame (ORF) and entire
promoter, enhancer, repressor/transactivator, origin of
replication, intron, operator, or any polynucleotide functional
group. Generally, a "defined sequences" for this purpose may be any
polynucleotide that a 15 base-polynucleotide sequence and
polynucleotide sequences of lengths between 15 bases and 15,000
bases (this invention specifically names every integer in between).
Considerations in choosing groupings of codons include types of
amino acids encoded by a degenerate mutagenic cassette.
[0342] In one exemplification a grouping of mutations that can be
introduced into a mutagenic cassette, this invention specifically
provides for degenerate codon substitutions (using degenerate
oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20 amino acids at each position and a
library of polypeptides encoded thereby.
[0343] Synthetic Ligation Reassembly (SLR)
[0344] The invention provides a non-stochastic gene modification
system termed "synthetic ligation reassembly," or simply "SLR," a
"directed evolution process," to generate polypeptides, e.g.,
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes or antibodies of the
invention, with new or altered properties. SLR is a method of
ligating oligonucleotide fragments together non-stochastically.
This method differs from stochastic oligonucleotide shuffling in
that the nucleic acid building blocks are not shuffled,
concatenated or chimerized randomly, but rather are assembled
non-stochastically. See, e.g., U.S. Pat. Nos. 6,773,900; 6,740,506;
6,713,282; 6,635,449; 6,605,449; 6,537,776.
[0345] In one aspect, SLR comprises the following steps: (a)
providing a template polynucleotide, wherein the template
polynucleotide comprises sequence encoding a homologous gene; (b)
providing a plurality of building block polynucleotides, wherein
the building block polynucleotides are designed to cross-over
reassemble with the template polynucleotide at a predetermined
sequence, and a building block polynucleotide comprises a sequence
that is a variant of the homologous gene and a sequence homologous
to the template polynucleotide flanking the variant sequence; (c)
combining a building block polynucleotide with a template
polynucleotide such that the building block polynucleotide
cross-over reassembles with the template polynucleotide to generate
polynucleotides comprising homologous gene sequence variations.
[0346] SLR does not depend on the presence of high levels of
homology between polynucleotides to be rearranged. Thus, this
method can be used to non-stochastically generate libraries (or
sets) of progeny molecules comprised of over 10.sup.100 different
chimeras. SLR can be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras. Thus, aspects of the
present invention include non-stochastic methods of producing a set
of finalized chimeric nucleic acid molecule shaving an overall
assembly order that is chosen by design. This method includes the
steps of generating by design a plurality of specific nucleic acid
building blocks having serviceable mutually compatible ligatable
ends, and assembling these nucleic acid building blocks, such that
a designed overall assembly order is achieved.
[0347] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, the overall
assembly order in which the nucleic acid building blocks can be
coupled is specified by the design of the ligatable ends. If more
than one assembly step is to be used, then the overall assembly
order in which the nucleic acid building blocks can be coupled is
also specified by the sequential order of the assembly step(s). In
one aspect, the annealed building pieces are treated with an
enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent
bonding of the building pieces.
[0348] In one aspect, the design of the oligonucleotide building
blocks is obtained by analyzing a set of progenitor nucleic acid
sequence templates that serve as a basis for producing a progeny
set of finalized chimeric polynucleotides. These parental
oligonucleotide templates thus serve as a source of sequence
information that aids in the design of the nucleic acid building
blocks that are to be mutagenized, e.g., chimerized or shuffled. In
one aspect of this method, the sequences of a plurality of parental
nucleic acid templates are aligned in order to select one or more
demarcation points. The demarcation points can be located at an
area of homology, and are comprised of one or more nucleotides.
These demarcation points are in one aspect shared by at least two
of the progenitor templates. The demarcation points can thereby be
used to delineate the boundaries of oligonucleotide building blocks
to be generated in order to rearrange the parental polynucleotides.
The demarcation points identified and selected in the progenitor
molecules serve as potential chimerization points in the assembly
of the final chimeric progeny molecules. A demarcation point can be
an area of homology (comprised of at least one homologous
nucleotide base) shared by at least two parental polynucleotide
sequences. Alternatively, a demarcation point can be an area of
homology that is shared by at least half of the parental
polynucleotide sequences, or, it can be an area of homology that is
shared by at least two thirds of the parental polynucleotide
sequences. Even more in one aspect a serviceable demarcation points
is an area of homology that is shared by at least three fourths of
the parental polynucleotide sequences, or, it can be shared by at
almost all of the parental polynucleotide sequences. In one aspect,
a demarcation point is an area of homology that is shared by all of
the parental polynucleotide sequences.
[0349] In one aspect, a ligation reassembly process is performed
exhaustively in order to generate an exhaustive library of progeny
chimeric polynucleotides. In other words, all possible ordered
combinations of the nucleic acid building blocks are represented in
the set of finalized chimeric nucleic acid molecules. At the same
time, in another aspect, the assembly order (i.e. the order of
assembly of each building block in the 5' to 3 sequence of each
finalized chimeric nucleic acid) in each combination is by design
(or non-stochastic) as described above. Because of the
non-stochastic nature of this invention, the possibility of
unwanted side products is greatly reduced.
[0350] In another aspect, the ligation reassembly method is
performed systematically. For example, the method is performed in
order to generate a systematically compartmentalized library of
progeny molecules, with compartments that can be screened
systematically, e.g. one by one. In other words this invention
provides that, through the selective and judicious use of specific
nucleic acid building blocks, coupled with the selective and
judicious use of sequentially stepped assembly reactions, a design
can be achieved where specific sets of progeny products are made in
each of several reaction vessels. This allows a systematic
examination and screening procedure to be performed. Thus, these
methods allow a potentially very large number of progeny molecules
to be examined systematically in smaller groups. Because of its
ability to perform chimerizations in a manner that is highly
flexible yet exhaustive and systematic as well, particularly when
there is a low level of homology among the progenitor molecules,
these methods provide for the generation of a library (or set)
comprised of a large number of progeny molecules. Because of the
non-stochastic nature of the instant ligation reassembly invention,
the progeny molecules generated in one aspect comprise a library of
finalized chimeric nucleic acid molecules having an overall
assembly order that is chosen by design. The saturation mutagenesis
and optimized directed evolution methods also can be used to
generate different progeny molecular species. It is appreciated
that the invention provides freedom of choice and control regarding
the selection of demarcation points, the size and number of the
nucleic acid building blocks, and the size and design of the
couplings. It is appreciated, furthermore, that the requirement for
intermolecular homology is highly relaxed for the operability of
this invention. In fact, demarcation points can even be chosen in
areas of little or no intermolecular homology. For example, because
of codon wobble, i.e. the degeneracy of codons, nucleotide
substitutions can be introduced into nucleic acid building blocks
without altering the amino acid originally encoded in the
corresponding progenitor template. Alternatively, a codon can be
altered such that the coding for an originally amino acid is
altered. This invention provides that such substitutions can be
introduced into the nucleic acid building block in order to
increase the incidence of intermolecular homologous demarcation
points and thus to allow an increased number of couplings to be
achieved among the building blocks, which in turn allows a greater
number of progeny chimeric molecules to be generated.
[0351] In one aspect, the present invention provides a
non-stochastic method termed synthetic gene reassembly, that is
somewhat related to stochastic shuffling, save that the nucleic
acid building blocks are not shuffled or concatenated or chimerized
randomly, but rather are assembled non-stochastically.
[0352] The synthetic gene reassembly method does not depend on the
presence of a high level of homology between polynucleotides to be
shuffled. The invention can be used to non-stochastically generate
libraries (or sets) of progeny molecules comprised of over
10.sup.100 different chimeras. Conceivably, synthetic gene
reassembly can even be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras.
[0353] Thus, in one aspect, the invention provides a non-stochastic
method of producing a set of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design, which method is comprised of the steps of generating by
design a plurality of specific nucleic acid building blocks having
serviceable mutually compatible ligatable ends and assembling these
nucleic acid building blocks, such that a designed overall assembly
order is achieved.
[0354] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, in one aspect,
the overall assembly order in which the nucleic acid building
blocks can be coupled is specified by the design of the ligatable
ends and, if more than one assembly step is to be used, then the
overall assembly order in which the nucleic acid building blocks
can be coupled is also specified by the sequential order of the
assembly step(s). In a one aspect of the invention, the annealed
building pieces are treated with an enzyme, such as a ligase (e.g.,
T4 DNA ligase) to achieve covalent bonding of the building
pieces.
[0355] In a another aspect, the design of nucleic acid building
blocks is obtained upon analysis of the sequences of a set of
progenitor nucleic acid templates that serve as a basis for
producing a progeny set of finalized chimeric nucleic acid
molecules. These progenitor nucleic acid templates thus serve as a
source of sequence information that aids in the design of the
nucleic acid building blocks that are to be mutagenized, i.e.
chimerized or shuffled.
[0356] In one exemplification, the invention provides for the
chimerization of a family of related genes and their encoded family
of related products. In a particular exemplification, the encoded
products are enzymes. The ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes of the present invention can be mutagenized in
accordance with the methods described herein.
[0357] Thus according to one aspect of the invention, the sequences
of a plurality of progenitor nucleic acid templates (e.g.,
polynucleotides of the invention) are aligned in order to select
one or more demarcation points, which demarcation points can be
located at an area of homology. The demarcation points can be used
to delineate the boundaries of nucleic acid building blocks to be
generated. Thus, the demarcation points identified and selected in
the progenitor molecules serve as potential chimerization points in
the assembly of the progeny molecules.
[0358] Typically a serviceable demarcation point is an area of
homology (comprised of at least one homologous nucleotide base)
shared by at least two progenitor templates, but the demarcation
point can be an area of homology that is shared by at least half of
the progenitor templates, at least two thirds of the progenitor
templates, at least three fourths of the progenitor templates and
in one aspect at almost all of the progenitor templates. Even more
in one aspect still a serviceable demarcation point is an area of
homology that is shared by all of the progenitor templates.
[0359] In a one aspect, the gene reassembly process is performed
exhaustively in order to generate an exhaustive library. In other
words, all possible ordered combinations of the nucleic acid
building blocks are represented in the set of finalized chimeric
nucleic acid molecules. At the same time, the assembly order (i.e.
the order of assembly of each building block in the 5' to 3
sequence of each finalized chimeric nucleic acid) in each
combination is by design (or non-stochastic). Because of the
non-stochastic nature of the method, the possibility of unwanted
side products is greatly reduced.
[0360] In another aspect, the method provides that the gene
reassembly process is performed systematically, for example to
generate a systematically compartmentalized library, with
compartments that can be screened systematically, e.g., one by one.
In other words the invention provides that, through the selective
and judicious use of specific nucleic acid building blocks, coupled
with the selective and judicious use of sequentially stepped
assembly reactions, an experimental design can be achieved where
specific sets of progeny products are made in each of several
reaction vessels. This allows a systematic examination and
screening procedure to be performed. Thus, it allows a potentially
very large number of progeny molecules to be examined
systematically in smaller groups.
[0361] Because of its ability to perform chimerizations in a manner
that is highly flexible yet exhaustive and systematic as well,
particularly when there is a low level of homology among the
progenitor molecules, the instant invention provides for the
generation of a library (or set) comprised of a large number of
progeny molecules. Because of the non-stochastic nature of the
instant gene reassembly invention, the progeny molecules generated
in one aspect comprise a library of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design. In a particularly aspect, such a generated library is
comprised of greater than 10.sup.3 to greater than 10.sup.1000
different progeny molecular species.
[0362] In one aspect, a set of finalized chimeric nucleic acid
molecules, produced as described is comprised of a polynucleotide
encoding a polypeptide. According to one aspect, this
polynucleotide is a gene, which may be a man-made gene. According
to another aspect, this polynucleotide is a gene pathway, which may
be a man-made gene pathway. The invention provides that one or more
man-made genes generated by the invention may be incorporated into
a man-made gene pathway, such as pathway operable in a eukaryotic
organism (including a plant).
[0363] In another exemplification, the synthetic nature of the step
in which the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.,
by mutagenesis) or in an in vivo process (e.g., by utilizing the
gene splicing ability of a host organism). It is appreciated that
in many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a serviceable demarcation point.
[0364] Thus, according to another aspect, the invention provides
that a nucleic acid building block can be used to introduce an
intron. Thus, the invention provides that functional introns may be
introduced into a man-made gene of the invention. The invention
also provides that functional introns may be introduced into a
man-made gene pathway of the invention. Accordingly, the invention
provides for the generation of a chimeric polynucleotide that is a
man-made gene containing one (or more) artificially introduced
intron(s).
[0365] Accordingly, the invention also provides for the generation
of a chimeric polynucleotide that is a man-made gene pathway
containing one (or more) artificially introduced intron(s). In one
aspect, the artificially introduced intron(s) are functional in one
or more host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene splicing.
The invention provides a process of producing man-made
intron-containing polynucleotides to be introduced into host
organisms for recombination and/or splicing.
[0366] A man-made gene produced using the invention can also serve
as a substrate for recombination with another nucleic acid.
Likewise, a man-made gene pathway produced using the invention can
also serve as a substrate for recombination with another nucleic
acid. In one aspect, the recombination is facilitated by, or occurs
at, areas of homology between the man-made, intron-containing gene
and a nucleic acid, which serves as a recombination partner. In one
aspect, the recombination partner may also be a nucleic acid
generated by the invention, including a man-made gene or a man-made
gene pathway. Recombination may be facilitated by or may occur at
areas of homology that exist at the one (or more) artificially
introduced intron(s) in the man-made gene.
[0367] The synthetic gene reassembly method of the invention
utilizes a plurality of nucleic acid building blocks, each of which
in one aspect has two ligatable ends. The two ligatable ends on
each nucleic acid building block may be two blunt ends (i.e. each
having an overhang of zero nucleotides), or in one aspect one blunt
end and one overhang, or more in one aspect still two
overhangs.
[0368] A useful overhang for this purpose may be a 3' overhang or a
5' overhang. Thus, a nucleic acid building block may have a 3'
overhang or alternatively a 5' overhang or alternatively two 3'
overhangs or alternatively two 5' overhangs. The overall order in
which the nucleic acid building blocks are assembled to form a
finalized chimeric nucleic acid molecule is determined by
purposeful experimental design and is not random.
[0369] In one aspect, a nucleic acid building block is generated by
chemical synthesis of two single-stranded nucleic acids (also
referred to as single-stranded oligos) and contacting them so as to
allow them to anneal to form a double-stranded nucleic acid
building block.
[0370] A double-stranded nucleic acid building block can be of
variable size. The sizes of these building blocks can be small or
large. Exemplary sizes for building block range from 1 base pair
(not including any overhangs) to 100,000 base pairs (not including
any overhangs). Other exemplary size ranges are also provided,
which have lower limits of from 1 bp to 10,000 bp (including every
integer value in between) and upper limits of from 2 bp to 100,000
bp (including every integer value in between).
[0371] Many methods exist by which a double-stranded nucleic acid
building block can be generated that is serviceable for the
invention; and these are known in the art and can be readily
performed by the skilled artisan.
[0372] According to one aspect, a double-stranded nucleic acid
building block is generated by first generating two single stranded
nucleic acids and allowing them to anneal to form a double-stranded
nucleic acid building block. The two strands of a double-stranded
nucleic acid building block may be complementary at every
nucleotide apart from any that form an overhang; thus containing no
mismatches, apart from any overhang(s). According to another
aspect, the two strands of a double-stranded nucleic acid building
block are complementary at fewer than every nucleotide apart from
any that form an overhang. Thus, according to this aspect, a
double-stranded nucleic acid building block can be used to
introduce codon degeneracy. In one aspect the codon degeneracy is
introduced using the site-saturation mutagenesis described herein,
using one or more N,N,G/T cassettes or alternatively using one or
more N,N,N cassettes.
[0373] The in vivo recombination method of the invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide.
[0374] The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, interleukin I, antibodies, tPA and growth
hormone. This approach may be used to generate proteins having
altered specificity or activity. The approach may also be useful
for the generation of hybrid nucleic acid sequences, for example,
promoter regions, introns, exons, enhancer sequences, 31
untranslated regions or 51 untranslated regions of genes. Thus this
approach may be used to generate genes having increased rates of
expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to
mutate ribozymes or aptamers.
[0375] In one aspect the invention described herein is directed to
the use of repeated cycles of reductive reassortment, recombination
and selection which allow for the directed molecular evolution of
highly complex linear sequences, such as DNA, RNA or proteins
thorough recombination.
[0376] Optimized Directed Evolution System
[0377] The invention provides a non-stochastic gene modification
system termed "optimized directed evolution system" to generate
polypeptides, e.g., ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzymes or antibodies of the invention, with new or altered
properties. Optimized directed evolution is directed to the use of
repeated cycles of reductive reassortment, recombination and
selection that allow for the directed molecular evolution of
nucleic acids through recombination. Optimized directed evolution
allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly
enriched for sequences that have a predetermined number of
crossover events.
[0378] A crossover event is a point in a chimeric sequence where a
shift in sequence occurs from one parental variant to another
parental variant. Such a point is normally at the juncture of where
oligonucleotides from two parents are ligated together to form a
single sequence. This method allows calculation of the correct
concentrations of oligonucleotide sequences so that the final
chimeric population of sequences is enriched for the chosen number
of crossover events. This provides more control over choosing
chimeric variants having a predetermined number of crossover
events.
[0379] In addition, this method provides a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. Previously, if one generated, for
example, 10.sup.13 chimeric molecules during a reaction, it would
be extremely difficult to test such a high number of chimeric
variants for a particular activity. Moreover, a significant portion
of the progeny population would have a very high number of
crossover events which resulted in proteins that were less likely
to have increased levels of a particular activity. By using these
methods, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0380] One method for creating a chimeric progeny polynucleotide
sequence is to create oligonucleotides corresponding to fragments
or portions of each parental sequence. Each oligonucleotide in one
aspect includes a unique region of overlap so that mixing the
oligonucleotides together results in a new variant that has each
oligonucleotide fragment assembled in the correct order.
Alternatively protocols for practicing these methods of the
invention can be found in U.S. Pat. Nos. 6,773,900; 6,740,506;
6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
[0381] The number of oligonucleotides generated for each parental
variant bears a relationship to the total number of resulting
crossovers in the chimeric molecule that is ultimately created. For
example, three parental nucleotide sequence variants might be
provided to undergo a ligation reaction in order to find a chimeric
variant having, for example, greater activity at high temperature.
As one example, a set of 50 oligonucleotide sequences can be
generated corresponding to each portions of each parental variant.
Accordingly, during the ligation reassembly process there could be
up to 50 crossover events within each of the chimeric sequences.
The probability that each of the generated chimeric polynucleotides
will contain oligonucleotides from each parental variant in
alternating order is very low. If each oligonucleotide fragment is
present in the ligation reaction in the same molar quantity it is
likely that in some positions oligonucleotides from the same
parental polynucleotide will ligate next to one another and thus
not result in a crossover event. If the concentration of each
oligonucleotide from each parent is kept constant during any
ligation step in this example, there is a 1/3 chance (assuming 3
parents) that an oligonucleotide from the same parental variant
will ligate within the chimeric sequence and produce no
crossover.
[0382] Accordingly, a probability density function (PDF) can be
determined to predict the population of crossover events that are
likely to occur during each step in a ligation reaction given a set
number of parental variants, a number of oligonucleotides
corresponding to each variant, and the concentrations of each
variant during each step in the ligation reaction. The statistics
and mathematics behind determining the PDF is described below. By
utilizing these methods, one can calculate such a probability
density function, and thus enrich the chimeric progeny population
for a predetermined number of crossover events resulting from a
particular ligation reaction. Moreover, a target number of
crossover events can be predetermined, and the system then
programmed to calculate the starting quantities of each parental
oligonucleotide during each step in the ligation reaction to result
in a probability density function that centers on the predetermined
number of crossover events. These methods are directed to the use
of repeated cycles of reductive reassortment, recombination and
selection that allow for the directed molecular evolution of a
nucleic acid encoding a polypeptide through recombination. This
system allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly
enriched for sequences that have a predetermined number of
crossover events. A crossover event is a point in a chimeric
sequence where a shift in sequence occurs from one parental variant
to another parental variant. Such a point is normally at the
juncture of where oligonucleotides from two parents are ligated
together to form a single sequence. The method allows calculation
of the correct concentrations of oligonucleotide sequences so that
the final chimeric population of sequences is enriched for the
chosen number of crossover events. This provides more control over
choosing chimeric variants having a predetermined number of
crossover events.
[0383] In addition, these methods provide a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. By using the methods described
herein, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0384] In one aspect, the method creates a chimeric progeny
polynucleotide sequence by creating oligonucleotides corresponding
to fragments or portions of each parental sequence. Each
oligonucleotide in one aspect includes a unique region of overlap
so that mixing the oligonucleotides together results in a new
variant that has each oligonucleotide fragment assembled in the
correct order. See also U.S. Pat. Nos. 6,773,900; 6,740,506;
6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
[0385] Determining Crossover Events
[0386] Aspects of the invention include a system and software that
receive a desired crossover probability density function (PDF), the
number of parent genes to be reassembled, and the number of
fragments in the reassembly as inputs. The output of this program
is a "fragment PDF" that can be used to determine a recipe for
producing reassembled genes, and the estimated crossover PDF of
those genes. The processing described herein is in one aspect
performed in MATLAB.TM. (The Mathworks, Natick, Mass.) a
programming language and development environment for technical
computing.
[0387] Iterative Processes
[0388] In practicing the invention, these processes can be
iteratively repeated. For example, a nucleic acid (or, the nucleic
acid) responsible for an altered or new ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme phenotype is identified,
re-isolated, again modified, re-tested for activity. This process
can be iteratively repeated until a desired phenotype is
engineered. For example, an entire biochemical anabolic or
catabolic pathway can be engineered into a cell, including, e.g.,
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme activity.
[0389] Similarly, if it is determined that a particular
oligonucleotide has no affect at all on the desired trait (e.g., a
new ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme phenotype), it
can be removed as a variable by synthesizing larger parental
oligonucleotides that include the sequence to be removed. Since
incorporating the sequence within a larger sequence prevents any
crossover events, there will no longer be any variation of this
sequence in the progeny polynucleotides. This iterative practice of
determining which oligonucleotides are most related to the desired
trait, and which are unrelated, allows more efficient exploration
all of the possible protein variants that might be provide a
particular trait or activity.
In Vivo Shuffling
[0390] In vivo shuffling of molecules is use in methods of the
invention that provide variants of polypeptides of the invention,
e.g., antibodies, ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes, and
the like. In vivo shuffling can be performed utilizing the natural
property of cells to recombine multimers. While recombination in
vivo has provided the major natural route to molecular diversity,
genetic recombination remains a relatively complex process that
involves 1) the recognition of homologies; 2) strand cleavage,
strand invasion, and metabolic steps leading to the production of
recombinant chiasma; and finally 3) the resolution of chiasma into
discrete recombined molecules. The formation of the chiasma
requires the recognition of homologous sequences.
[0391] In another aspect, the invention includes a method for
producing a hybrid polynucleotide from at least a first
polynucleotide and a second polynucleotide. The invention can be
used to produce a hybrid polynucleotide by introducing at least a
first polynucleotide and a second polynucleotide (e.g., one, or
both, being an exemplary ammonia lyase, e.g., phenylalanine
ammoniac lyase, histidine ammonia lyase and/or tyrosine ammonia
lyase, enzyme-encoding sequence of the invention) which share at
least one region of partial sequence homology into a suitable host
cell. The regions of partial sequence homology promote processes
which result in sequence reorganization producing a hybrid
polynucleotide. The term "hybrid polynucleotide", as used herein,
is any nucleotide sequence which results from the method of the
present invention and contains sequence from at least two original
polynucleotide sequences. Such hybrid polynucleotides can result
from intermolecular recombination events which promote sequence
integration between DNA molecules. In addition, such hybrid
polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[0392] In vivo reassortment is focused on "inter-molecular"
processes collectively referred to as "recombination" which in
bacteria, is generally viewed as a "RecA-dependent" phenomenon. The
invention can rely on recombination processes of a host cell to
recombine and re-assort sequences, or the cells' ability to mediate
reductive processes to decrease the complexity of quasi-repeated
sequences in the cell by deletion. This process of "reductive
reassortment" occurs by an "intra-molecular", RecA-independent
process.
[0393] Therefore, in another aspect of the invention, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs
containing consecutive sequences (original encoding sequences),
their insertion into an appropriate vector and their subsequent
introduction into an appropriate host cell. The reassortment of the
individual molecular identities occurs by combinatorial processes
between the consecutive sequences in the construct possessing
regions of homology, or between quasi-repeated units. The
reassortment process recombines and/or reduces the complexity and
extent of the repeated sequences and results in the production of
novel molecular species. Various treatments may be applied to
enhance the rate of reassortment. These could include treatment
with ultra-violet light, or DNA damaging chemicals and/or the use
of host cell lines displaying enhanced levels of "genetic
instability". Thus the reassortment process may involve homologous
recombination or the natural property of quasi-repeated sequences
to direct their own evolution.
[0394] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In the present invention, "quasi-repeats" are
repeats that are not restricted to their original unit structure.
Quasi-repeated units can be presented as an array of sequences in a
construct; consecutive units of similar sequences. Once ligated,
the junctions between the consecutive sequences become essentially
invisible and the quasi-repetitive nature of the resulting
construct is now continuous at the molecular level. The deletion
process the cell performs to reduce the complexity of the resulting
construct operates between the quasi-repeated sequences. The
quasi-repeated units provide a practically limitless repertoire of
templates upon which slippage events can occur. The constructs
containing the quasi-repeats thus effectively provide sufficient
molecular elasticity that deletion (and potentially insertion)
events can occur virtually anywhere within the quasi-repetitive
units.
[0395] When the quasi-repeated sequences are all ligated in the
same orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, it is preferable with the present method that the sequences
are in the same orientation. Random orientation of quasi-repeated
sequences will result in the loss of reassortment efficiency, while
consistent orientation of the sequences will offer the highest
efficiency. However, while having fewer of the contiguous sequences
in the same orientation decreases the efficiency, it may still
provide sufficient elasticity for the effective recovery of novel
molecules. Constructs can be made with the quasi-repeated sequences
in the same orientation to allow higher efficiency.
[0396] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following: [0397]
a) Primers that include a poly-A head and poly-T tail which when
made single-stranded would provide orientation can be utilized.
This is accomplished by having the first few bases of the primers
made from RNA and hence easily removed RNaseH. [0398] b) Primers
that include unique restriction cleavage sites can be utilized.
Multiple sites, a battery of unique sequences and repeated
synthesis and ligation steps would be required. [0399] c) The inner
few bases of the primer could be thiolated and an exonuclease used
to produce properly tailed molecules.
[0400] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced repetitive index
(RI). The re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The
recovery of cloning vectors with reduced RI can be affected by:
[0401] 1) The use of vectors only stably maintained when the
construct is reduced in complexity. [0402] 2) The physical recovery
of shortened vectors by physical procedures. In this case, the
cloning vector would be recovered using standard plasmid isolation
procedures and size fractionated on either an agarose gel, or
column with a low molecular weight cut off utilizing standard
procedures. [0403] 3) The recovery of vectors containing
interrupted genes which can be selected when insert size decreases.
[0404] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0405] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, while the examples illustrated below demonstrate the
reassortment of nearly identical original encoding sequences
(quasi-repeats), this process is not limited to such nearly
identical repeats.
[0406] The following example demonstrates a method of the
invention. Encoding nucleic acid sequences (quasi-repeats) derived
from three (3) unique species are described. Each sequence encodes
a protein with a distinct set of properties. Each of the sequences
differs by a single or a few base pairs at a unique position in the
sequence. The quasi-repeated sequences are separately or
collectively amplified and ligated into random assemblies such that
all possible permutations and combinations are available in the
population of ligated molecules. The number of quasi-repeat units
can be controlled by the assembly conditions. The average number of
quasi-repeated units in a construct is defined as the repetitive
index (RI).
[0407] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassortment
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in RI is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[0408] Optionally, the method comprises the additional step of
screening the library members of the shuffled pool to identify
individual shuffled library members having the ability to bind or
otherwise interact, or catalyze a particular reaction (e.g., such
as catalytic domain of an enzyme) with a predetermined
macromolecule, such as for example a proteinaceous receptor, an
oligosaccharide, virion, or other predetermined compound or
structure.
[0409] The polypeptides that are identified from such libraries can
be used for therapeutic, diagnostic, research and related purposes
(e.g., catalysts, solutes for increasing osmolarity of an aqueous
solution and the like) and/or can be subjected to one or more
additional cycles of shuffling and/or selection.
[0410] In another aspect, it is envisioned that prior to or during
recombination or reassortment, polynucleotides generated by the
method of the invention can be subjected to agents or processes
which promote the introduction of mutations into the original
polynucleotides. The introduction of such mutations would increase
the diversity of resulting hybrid polynucleotides and polypeptides
encoded therefrom. The agents or processes which promote
mutagenesis can include, but are not limited to: (+)-CC-1065, or a
synthetic analog such as (+)-CC-1065-(N3-Adenine (See Sun and
Hurley, (1992); an N-acetylated or deacetylated
4'-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA
synthesis (See, for example, van de Poll et al. (1992)); or a
N-acetylated or deacetylated 4-aminobiphenyl adduct capable of
inhibiting DNA synthesis (See also, van de Poll et al. (1992), pp.
751-758); trivalent chromium, a trivalent chromium salt, a
polycyclic aromatic hydrocarbon (PAH) DNA adduct capable of
inhibiting DNA replication, such as
7-bromomethyl-benz[.alpha.]anthracene ("BMA"),
tris(2,3-dibromopropyl)phosphate ("Tris-BP"),
1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[.alpha.]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a
platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline ("N-hydroxy-IQ")
and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine
("N-hydroxy-PhIP"). Exemplary means for slowing or halting PCR
amplification consist of UV light (+)-CC-1065 and
(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA
adducts or polynucleotides comprising the DNA adducts from the
polynucleotides or polynucleotides pool, which can be released or
removed by a process including heating the solution comprising the
polynucleotides prior to further processing.
[0411] In another aspect the invention is directed to a method of
producing recombinant proteins having biological activity by
treating a sample comprising double-stranded template
polynucleotides encoding a wild-type protein under conditions
according to the invention which provide for the production of
hybrid or re-assorted polynucleotides.
[0412] Producing Sequence Variants
[0413] The invention also provides additional methods for making
sequence variants of the nucleic acid (e.g., ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme) sequences of the invention. The
invention also provides additional methods for isolating ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzymes using the nucleic acids and
polypeptides of the invention. In one aspect, the invention
provides for variants of an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme coding sequence (e.g., a gene, cDNA or message) of the
invention, which can be altered by any means, including, e.g.,
random or stochastic methods, or, non-stochastic, or "directed
evolution," methods, as described above.
[0414] The isolated variants may be naturally occurring. Variant
can also be created in vitro. Variants may be created using genetic
engineering techniques such as site directed mutagenesis, random
chemical mutagenesis, Exonuclease III deletion procedures, and
standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives may be created using chemical
synthesis or modification procedures. Other methods of making
variants are also familiar to those skilled in the art. These
include procedures in which nucleic acid sequences obtained from
natural isolates are modified to generate nucleic acids which
encode polypeptides having characteristics which enhance their
value in industrial or laboratory applications. In such procedures,
a large number of variant sequences having one or more nucleotide
differences with respect to the sequence obtained from the natural
isolate are generated and characterized. These nucleotide
differences can result in amino acid changes with respect to the
polypeptides encoded by the nucleic acids from the natural
isolates.
[0415] For example, variants may be created using error prone PCR.
In error prone PCR, PCR is performed under conditions where the
copying fidelity of the DNA polymerase is low, such that a high
rate of point mutations is obtained along the entire length of the
PCR product. Error prone PCR is described, e.g., in Leung (1989)
Technique 1:11-15) and Caldwell (1992) PCR Methods Applic. 2:28-33.
Briefly, in such procedures, nucleic acids to be mutagenized are
mixed with PCR primers, reaction buffer, MgCl.sub.2, MnCl.sub.2,
Taq polymerase and an appropriate concentration of dNTPs for
achieving a high rate of point mutation along the entire length of
the PCR product. For example, the reaction may be performed using
20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR
primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH
8.3) and 0.01% gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units
of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM
dTTP. PCR may be performed for 30 cycles of 94.degree. C. for 1
min, 45.degree. C. for 1 min, and 72.degree. C. for 1 min. However,
it will be appreciated that these parameters may be varied as
appropriate. The mutagenized nucleic acids are cloned into an
appropriate vector and the activities of the polypeptides encoded
by the mutagenized nucleic acids are evaluated.
[0416] Variants may also be created using oligonucleotide directed
mutagenesis to generate site-specific mutations in any cloned DNA
of interest. Oligonucleotide mutagenesis is described, e.g., in
Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such
procedures a plurality of double stranded oligonucleotides bearing
one or more mutations to be introduced into the cloned DNA are
synthesized and inserted into the cloned DNA to be mutagenized.
Clones containing the mutagenized DNA are recovered and the
activities of the polypeptides they encode are assessed.
[0417] Another method for generating variants is assembly PCR.
Assembly PCR involves the assembly of a PCR product from a mixture
of small DNA fragments. A large number of different PCR reactions
occur in parallel in the same vial, with the products of one
reaction priming the products of another reaction. Assembly PCR is
described in, e.g., U.S. Pat. No. 5,965,408.
[0418] Still another method of generating variants is sexual PCR
mutagenesis. In sexual
[0419] PCR mutagenesis, forced homologous recombination occurs
between DNA molecules of different but highly related DNA sequence
in vitro, as a result of random fragmentation of the DNA molecule
based on sequence homology, followed by fixation of the crossover
by primer extension in a PCR reaction. Sexual PCR mutagenesis is
described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751. Briefly, in such procedures a plurality of nucleic
acids to be recombined are digested with DNase to generate
fragments having an average size of 50-200 nucleotides. Fragments
of the desired average size are purified and resuspended in a PCR
mixture. PCR is conducted under conditions which facilitate
recombination between the nucleic acid fragments. For example, PCR
may be performed by resuspending the purified fragments at a
concentration of 10-30 ng/.mu.l in a solution of 0.2 mM of each
dNTP, 2.2 mM MgCl.sub.2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and
0.1% Triton X-100. 2.5 units of Taq polymerase per 100:1 of
reaction mixture is added and PCR is performed using the following
regime: 94.degree. C. for 60 seconds, 94.degree. C. for 30 seconds,
50-55.degree. C. for 30 seconds, 72.degree. C. for 30 seconds
(30-45 times) and 72.degree. C. for 5 minutes. However, it will be
appreciated that these parameters may be varied as appropriate. In
some aspects, oligonucleotides may be included in the PCR
reactions. In other aspects, the Klenow fragment of DNA polymerase
I may be used in a first set of PCR reactions and Taq polymerase
may be used in a subsequent set of PCR reactions. Recombinant
sequences are isolated and the activities of the polypeptides they
encode are assessed.
[0420] Variants may also be created by in vivo mutagenesis. In some
aspects, random mutations in a sequence of interest are generated
by propagating the sequence of interest in a bacterial strain, such
as an E. coli strain, which carries mutations in one or more of the
DNA repair pathways. Such "mutator" strains have a higher random
mutation rate than that of a wild-type parent. Propagating the DNA
in one of these strains will eventually generate random mutations
within the DNA. Mutator strains suitable for use for in vivo
mutagenesis are described in PCT Publication No. WO 91/16427,
published Oct. 31, 1991, entitled "Methods for Phenotype Creation
from Multiple Gene Populations".
[0421] Variants may also be generated using cassette mutagenesis.
In cassette mutagenesis a small region of a double stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains completely and/or partially randomized native
sequence.
[0422] Recursive ensemble mutagenesis may also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (protein mutagenesis) developed to produce
diverse populations of phenotypically related mutants whose members
differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described, e.g., in
Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
[0423] In some aspects, variants are created using exponential
ensemble mutagenesis. Exponential ensemble mutagenesis is a process
for generating combinatorial libraries with a high percentage of
unique and functional mutants, wherein small groups of residues are
randomized in parallel to identify, at each altered position, amino
acids which lead to functional proteins. Exponential ensemble
mutagenesis is described, e.g., in Delegrave (1993) Biotechnology
Res. 11:1548-1552. Random and site-directed mutagenesis are
described, e.g., in Arnold (1993) Current Opinion in Biotechnology
4:450-455.
[0424] In some aspects, the variants are created using shuffling
procedures wherein portions of a plurality of nucleic acids which
encode distinct polypeptides are fused together to create chimeric
nucleic acid sequences which encode chimeric polypeptides as
described in U.S. Pat. No. 5,965,408, filed Jul. 9, 1996, entitled,
"Method of DNA Reassembly by Interrupting Synthesis" and U.S. Pat.
No. 5,939,250, filed May 22, 1996, entitled, "Production of Enzymes
Having Desired Activities by Mutagenesis.
[0425] The variants of the polypeptides of the invention may be
variants in which one or more of the amino acid residues of the
polypeptides of the sequences of the invention are substituted with
a conserved or non-conserved amino acid residue (in one aspect a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code.
[0426] Conservative substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the following replacements: replacements of an aliphatic amino acid
such as Alanine, Valine, Leucine and Isoleucine with another
aliphatic amino acid; replacement of a Serine with a Threonine or
vice versa; replacement of an acidic residue such as Aspartic acid
and Glutamic acid with another acidic residue; replacement of a
residue bearing an amide group, such as Asparagine and Glutamine,
with another residue bearing an amide group; exchange of a basic
residue such as Lysine and Arginine with another basic residue; and
replacement of an aromatic residue such as Phenylalanine, Tyrosine
with another aromatic residue. Other variants are those in which
one or more of the amino acid residues of a polypeptide of the
invention includes a substituent group.
[0427] In one aspect, a conservative substitution is a substitution
of one amino acid for another amino acid in a polypeptide, which
substitution has little to no impact on the structure and/or
activity (including binding and/or enzymatic activity) of the
polypeptide. The substitution is considered conservative
independent of whether the exchanged amino acids appear
structurally or functionally similar. For example, ideally, a lyase
polypeptide including one or more conservative substitutions
retains lyase activity. A polypeptide can be produced to contain
one or more conservative substitutions by manipulating the
nucleotide sequence that encodes that polypeptide using, for
example, standard procedures such as site-directed mutagenesis or
PCR or other methods known to those in the art.
[0428] Non-limiting examples of amino acids which may be
substituted for an original amino acid in a protein and which may
be regarded as conservative substitutions if there is little to no
impact on the activity of the polypeptide include: Ala substituted
with ser or thr; arg substituted with gln, his, or lys; asn
substituted with glu, gln, lys, his, asp; asp substituted with asn,
glu, or gln; cys substituted with ser or ala; gln substituted with
asn, glu, lys, his, asp, or arg; glu substituted with asn, gln lys,
or asp; gly substituted with pro; his substituted with asn, lys,
gln, arg, tyr; ile substituted with leu, met, val, phe; leu
substituted with ile, met, val, phe; lys substituted with asn, glu,
gln, his, arg; met substituted with ile, leu, val, phe; phe
substituted with trp, tyr, met, ile, or leu; ser substituted with
thr, ala; thr substituted with ser or ala; trp substituted with
phe, tyr; tyr substituted with his, phe, or trp; and val
substituted with met, ile, leu.
[0429] Further information about conservative substitutions can be
found in, among other locations, Ben-Bassat et al., (J. Bacteriol.
169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989),
Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al.,
(Bio/Technology 6:1321-5, 1988), WO 00/67796 (Curd et al.) and in
standard textbooks of genetics and molecular biology.
[0430] Still other variants are those in which the polypeptide is
associated with another compound, such as a compound to increase
the half-life of the polypeptide (for example, polyethylene
glycol).
[0431] Additional variants are those in which additional amino
acids are fused to the polypeptide, such as a leader sequence, a
secretory sequence, a proprotein sequence or a sequence which
facilitates purification, enrichment, or stabilization of the
polypeptide.
[0432] In some aspects, the fragments, derivatives and analogs
retain the same biological function or activity as the polypeptides
of the invention. In other aspects, the fragment, derivative, or
analog includes a proprotein, such that the fragment, derivative,
or analog can be activated by cleavage of the proprotein portion to
produce an active polypeptide.
[0433] Optimizing Codons to Achieve High Levels of Protein
Expression in Host Cells
[0434] The invention provides methods for modifying ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase, enzyme-encoding nucleic acids to modify
codon usage. In one aspect, the invention provides methods for
modifying codons in a nucleic acid encoding an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme to increase or decrease its
expression in a host cell. The invention also provides nucleic
acids encoding an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
modified to increase its expression in a host cell, ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme so modified, and methods of making
the modified ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes. The
method comprises identifying a "non-preferred" or a "less
preferred" codon in ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase,
enzyme-encoding nucleic acid and replacing one or more of these
non-preferred or less preferred codons with a "preferred codon"
encoding the same amino acid as the replaced codon and at least one
non-preferred or less preferred codon in the nucleic acid has been
replaced by a preferred codon encoding the same amino acid. A
preferred codon is a codon over-represented in coding sequences in
genes in the host cell and a non-preferred or less preferred codon
is a codon under-represented in coding sequences in genes in the
host cell.
[0435] Host cells for expressing the nucleic acids, expression
cassettes and vectors of the invention include bacteria, yeast,
fungi, plant cells, insect cells and mammalian cells. Thus, the
invention provides methods for optimizing codon usage in all of
these cells, codon-altered nucleic acids and polypeptides made by
the codon-altered nucleic acids. Exemplary host cells include gram
negative bacteria, such as Escherichia coli; gram positive
bacteria, such as Streptomyces sp., Lactobacillus gasseri,
Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis,
Bacillus cereus. Exemplary host cells also include eukaryotic
organisms, e.g., various yeast, such as Saccharomyces sp.,
including Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,
Aspergillus niger, and mammalian cells and cell lines and insect
cells and cell lines. Thus, the invention also includes nucleic
acids and polypeptides optimized for expression in these organisms
and species.
[0436] For example, the codons of a nucleic acid encoding an
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme isolated from a
bacterial cell are modified such that the nucleic acid is optimally
expressed in a bacterial cell different from the bacteria from
which the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme was
derived, a yeast, a fungi, a plant cell, an insect cell or a
mammalian cell. Methods for optimizing codons are well known in the
art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.
Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;
Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001)
Infect. Immun 69:7250-7253, describing optimizing codons in mouse
systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,
describing optimizing codons in yeast; Feng (2000) Biochemistry
39:15399-15409, describing optimizing codons in E. coli; Humphreys
(2000) Protein Expr. Purif. 20:252-264, describing optimizing codon
usage that affects secretion in E. coli.
Transgenic Non-Human Animals
[0437] The invention provides transgenic non-human animals
comprising a nucleic acid, a polypeptide (e.g., an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme), an expression cassette or vector
or a transfected or transformed cell of the invention. The
invention also provides methods of making and using these
transgenic non-human animals.
[0438] The transgenic non-human animals can be, e.g., goats,
rabbits, sheep, pigs (including all swine, hogs and related
animals), cows, rats and mice, comprising the nucleic acids of the
invention. These animals can be used, e.g., as in vivo models to
study ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme activity, or,
as models to screen for agents that change the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity in vivo. The coding
sequences for the polypeptides to be expressed in the transgenic
non-human animals can be designed to be constitutive, or, under the
control of tissue-specific, developmental-specific or inducible
transcriptional regulatory factors. Transgenic non-human animals
can be designed and generated using any method known in the art;
see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952;
6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070;
5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571,
describing making and using transformed cells and eggs and
transgenic mice, rats, rabbits, sheep, pigs and cows. See also,
e.g., Pollock (1999) J. Immunol. Methods 231:147-157, describing
the production of recombinant proteins in the milk of transgenic
dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461,
demonstrating the production of transgenic goats. U.S. Pat. No.
6,211,428, describes making and using transgenic non-human mammals
which express in their brains a nucleic acid construct comprising a
DNA sequence. U.S. Pat. No. 5,387,742, describes injecting cloned
recombinant or synthetic DNA sequences into fertilized mouse eggs,
implanting the injected eggs in pseudo-pregnant females, and
growing to term transgenic mice. U.S. Pat. No. 6,187,992, describes
making and using a transgenic mouse.
[0439] "Knockout animals" can also be used to practice the methods
of the invention. For example, in one aspect, the transgenic or
modified animals of the invention comprise a "knockout animal,"
e.g., a "knockout mouse," engineered not to express an endogenous
gene, which is replaced with a gene expressing an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme of the invention, or, a fusion
protein comprising an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
of the invention.
Transgenic Plants and Seeds
[0440] The invention provides transgenic plants and seeds
comprising a nucleic acid, a polypeptide (e.g., an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme), an expression cassette or vector
or a transfected or transformed cell of the invention. The
invention also provides plant products, e.g., oils, seeds, leaves,
extracts and the like, comprising a nucleic acid and/or a
polypeptide (e.g., an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzyme) of the invention. The transgenic plant can be
dicotyledonous (a dicot) or monocotyledonous (a monocot). The
invention also provides methods of making and using these
transgenic plants and seeds. The transgenic plant or plant cell
expressing a polypeptide of the present invention may be
constructed in accordance with any method known in the art. See,
for example, U.S. Pat. No. 6,309,872.
[0441] Nucleic acids and expression constructs of the invention can
be introduced into a plant cell by any means. For example, nucleic
acids or expression constructs can be introduced into the genome of
a desired plant host, or, the nucleic acids or expression
constructs can be episomes. Introduction into the genome of a
desired plant can be such that the host's ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme production is regulated by
endogenous transcriptional or translational control elements. The
invention also provides "knockout plants" where insertion of gene
sequence by, e.g., homologous recombination, has disrupted the
expression of the endogenous gene. Means to generate "knockout"
plants are well-known in the art, see, e.g., Strepp (1998) Proc
Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J 7:359-365.
See discussion on transgenic plants, below.
[0442] The nucleic acids of the invention can be used to confer
desired traits on essentially any plant, e.g., on starch-producing
plants, such as potato, wheat, rice, barley, and the like. Nucleic
acids of the invention can be used to manipulate metabolic pathways
of a plant in order to optimize or alter host's expression of
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme. The can change ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme activity in a plant.
Alternatively, an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme of the
invention can be used in production of a transgenic plant to
produce a compound not naturally produced by that plant. This can
lower production costs or create a novel product.
[0443] In one aspect, the first step in production of a transgenic
plant involves making an expression construct for expression in a
plant cell. These techniques are well known in the art. They can
include selecting and cloning a promoter, a coding sequence for
facilitating efficient binding of ribosomes to mRNA and selecting
the appropriate gene terminator sequences. One exemplary
constitutive promoter is CaMV35S, from the cauliflower mosaic
virus, which generally results in a high degree of expression in
plants. Other promoters are more specific and respond to cues in
the plant's internal or external environment. An exemplary
light-inducible promoter is the promoter from the cab gene,
encoding the major chlorophyll a/b binding protein.
[0444] In one aspect, the nucleic acid is modified to achieve
greater expression in a plant cell. For example, a sequence of the
invention is likely to have a higher percentage of A-T nucleotide
pairs compared to that seen in a plant, some of which prefer G-C
nucleotide pairs. Therefore, A-T nucleotides in the coding sequence
can be substituted with G-C nucleotides without significantly
changing the amino acid sequence to enhance production of the gene
product in plant cells.
[0445] Selectable marker gene can be added to the gene construct in
order to identify plant cells or tissues that have successfully
integrated the transgene. This may be necessary because achieving
incorporation and expression of genes in plant cells is a rare
event, occurring in just a few percent of the targeted tissues or
cells. Selectable marker genes encode proteins that provide
resistance to agents that are normally toxic to plants, such as
antibiotics or herbicides. Only plant cells that have integrated
the selectable marker gene will survive when grown on a medium
containing the appropriate antibiotic or herbicide. As for other
inserted genes, marker genes also require promoter and termination
sequences for proper function.
[0446] In one aspect, making transgenic plants or seeds comprises
incorporating sequences of the invention and, optionally, marker
genes into a target expression construct (e.g., a plasmid), along
with positioning of the promoter and the terminator sequences. This
can involve transferring the modified gene into the plant through a
suitable method. For example, a construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the constructs can be introduced directly to plant
tissue using ballistic methods, such as DNA particle bombardment.
For example, see, e.g., Christou (1997) Plant Mol. Biol.
35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987)
Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,
discussing use of particle bombardment to introduce transgenes into
wheat; and Adam (1997) supra, for use of particle bombardment to
introduce YACs into plant cells. For example, Rinehart (1997)
supra, used particle bombardment to generate transgenic cotton
plants. Apparatus for accelerating particles is described U.S. Pat.
No. 5,015,580; and, the commercially available BioRad (Biolistics)
PDS-2000 particle acceleration instrument; see also, John, U.S.
Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describing
particle-mediated transformation of gymnosperms.
[0447] In one aspect, protoplasts can be immobilized and injected
with a nucleic acids, e.g., an expression construct. Although plant
regeneration from protoplasts is not easy with cereals, plant
regeneration is possible in legumes using somatic embryogenesis
from protoplast derived callus. Organized tissues can be
transformed with naked DNA using gene gun technique, where DNA is
coated on tungsten microprojectiles, shot 1/100th the size of
cells, which carry the DNA deep into cells and organelles.
Transformed tissue is then induced to regenerate, usually by
somatic embryogenesis. This technique has been successful in
several cereal species including maize and rice.
[0448] Nucleic acids, e.g., expression constructs, can also be
introduced in to plant cells using recombinant viruses. Plant cells
can be transformed using viral vectors, such as, e.g., tobacco
mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol.
33:989-999), see Porta (1996) "Use of viral replicons for the
expression of genes in plants," Mol. Biotechnol. 5:209-221.
[0449] Alternatively, nucleic acids, e.g., an expression construct,
can be combined with suitable T-DNA flanking regions and introduced
into a conventional Agrobacterium tumefaciens host vector. The
virulence functions of the Agrobacterium tumefaciens host will
direct the insertion of the construct and adjacent marker into the
plant cell DNA when the cell is infected by the bacteria.
Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary vectors, are well described
in the scientific literature. See, e.g., Horsch (1984) Science
233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803
(1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag,
Berlin 1995). The DNA in an A. tumefaciens cell is contained in the
bacterial chromosome as well as in another structure known as a Ti
(tumor-inducing) plasmid. The Ti plasmid contains a stretch of DNA
termed T-DNA (.about.20 kb long) that is transferred to the plant
cell in the infection process and a series of vir (virulence) genes
that direct the infection process. A. tumefaciens can only infect a
plant through wounds: when a plant root or stem is wounded it gives
off certain chemical signals, in response to which, the vir genes
of A. tumefaciens become activated and direct a series of events
necessary for the transfer of the T-DNA from the Ti plasmid to the
plant's chromosome. The T-DNA then enters the plant cell through
the wound. One speculation is that the T-DNA waits until the plant
DNA is being replicated or transcribed, then inserts itself into
the exposed plant DNA. In order to use A. tumefaciens as a
transgene vector, the tumor-inducing section of T-DNA have to be
removed, while retaining the T-DNA border regions and the vir
genes. The transgene is then inserted between the T-DNA border
regions, where it is transferred to the plant cell and becomes
integrated into the plant's chromosomes.
[0450] The invention provides for the transformation of
monocotyledonous plants using the nucleic acids of the invention,
including important cereals, see Hiei (1997) Plant Mol. Biol.
35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley
(1983) Proc. Natl. Acad. Sci. USA 80:4803; Thykjaer (1997) supra;
Park (1996) Plant Mol. Biol. 32:1135-1148, discussing T-DNA
integration into genomic DNA. See also D'Halluin, U.S. Pat. No.
5,712,135, describing a process for the stable integration of a DNA
comprising a gene that is functional in a cell of a cereal, or
other monocotyledonous plant.
[0451] In one aspect, the third step can involve selection and
regeneration of whole plants capable of transmitting the
incorporated target gene to the next generation. Such regeneration
techniques rely on manipulation of certain phytohormones in a
tissue culture growth medium, typically relying on a biocide and/or
herbicide marker that has been introduced together with the desired
nucleotide sequences. Plant regeneration from cultured protoplasts
is described in Evans et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing
Company, New York, 1983; and Binding, Regeneration of Plants, Plant
Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration
can also be obtained from plant callus, explants, organs, or parts
thereof. Such regeneration techniques are described generally in
Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole
plants from transgenic tissues such as immature embryos, they can
be grown under controlled environmental conditions in a series of
media containing nutrients and hormones, a process known as tissue
culture. Once whole plants are generated and produce seed,
evaluation of the progeny begins.
[0452] After the expression cassette is stably incorporated in
transgenic plants, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed. Since transgenic
expression of the nucleic acids of the invention leads to
phenotypic changes, plants comprising the recombinant nucleic acids
of the invention can be sexually crossed with a second plant to
obtain a final product. Thus, the seed of the invention can be
derived from a cross between two transgenic plants of the
invention, or a cross between a plant of the invention and another
plant. The desired effects (e.g., expression of the polypeptides of
the invention to produce a plant in which flowering behavior is
altered) can be enhanced when both parental plants express the
polypeptides (e.g., an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzyme) of the invention. The desired effects can be passed to
future plant generations by standard propagation means.
[0453] The nucleic acids and polypeptides of the invention are
expressed in or inserted in any plant or seed. Transgenic plants of
the invention can be dicotyledonous or monocotyledonous. Examples
of monocot transgenic plants of the invention are grasses, such as
meadow grass (blue grass, Poa), forage grass such as festuca,
lolium, temperate grass, such as Agrostis, and cereals, e.g.,
wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples
of dicot transgenic plants of the invention are tobacco, legumes,
such as lupins, potato, sugar beet, pea, bean and soybean, and
cruciferous plants (family Brassicaceae), such as cauliflower, rape
seed, and the closely related model organism Arabidopsis thaliana.
Thus, the transgenic plants and seeds of the invention include a
broad range of plants, including, but not limited to, species from
the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica,
Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis,
Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,
Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum,
Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago,
Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus,
Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,
Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella,
Triticum, Vicia, Vitis, Vigna, and Zea.
[0454] In alternative embodiments, the nucleic acids of the
invention are expressed in plants which contain fiber cells,
including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra),
desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp,
roselle, jute, sisal abaca and flax. In alternative embodiments,
the transgenic plants of the invention can be members of the genus
Gossypium, including members of any Gossypium species, such as G.
arboreum; G. herbaceum, G. barbadense, and G. hirsutum.
[0455] The invention also provides for transgenic plants to be used
for producing large amounts of the polypeptides (e.g., an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme or antibody) of the
invention. For example, see Palmgren (1997) Trends Genet. 13:348;
Chong (1997) Transgenic Res. 6:289-296 (producing human milk
protein beta-casein in transgenic potato plants using an
auxin-inducible, bidirectional mannopine synthase (mas1',2')
promoter with Agrobacterium tumefaciens-mediated leaf disc
transformation methods).
[0456] Using known procedures, one of skill can screen for plants
of the invention by detecting the increase or decrease of transgene
mRNA or protein in transgenic plants. Means for detecting and
quantitation of mRNAs or proteins are well known in the art.
Polypeptides and Peptides
[0457] In one aspect, the invention provides isolated, synthetic or
recombinant polypeptides having a sequence identity (e.g., at least
about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more, or complete (100%) sequence identity, or homology) to an
exemplary sequence of the invention, including all even-numbered
SEQ ID NO:s between SEQ ID NO:2 and SEQ ID NO:102). The percent
sequence identity can be over the full length of the polypeptide,
or, the identity can be over a region of at least about 50, 60, 70,
80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700 or more residues. In one aspect, the polypeptides of the
invention have a lyase activity, e.g., at least one activity, such
as having at least an ammonia lyase activity, as set forth in Table
1 (FIG. 8), Table 2 (FIG. 9), and Table 3 (below), and the
explanation as discussed above, where it is noted that Tables 1 and
2, presented as FIG. 8 and FIG. 9, respectively, detail exemplary
activities of polypeptides of the invention; noting that each
polypeptide of the invention can have more than one specific
enzymatic activity. See also explanation above for guide to reading
Table 3.
TABLE-US-00002 TABLE 3 Predicted EC Top hit NR, % protein SEQ ID
NO: Activity number Source NR top hit annotation Accession No.
identity 1, 2 Ammonia-lyase 4.3.1.3 Unknown histidine ammonia-lyase
37198001 66 [Vibrio vulnificus YJ016] 3, 4 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 98 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 5, 6 Ammonia-lyase 4.3.1.3 Unknown
Histidine ammonia-lyase 56178255 62 [Idiomarina Ioihiensis L2TR] 7,
8 Ammonia-lyase 4.3.1.3 Unknown Pal/histidase family protein
68348275 92 [Pseudomonas fluorescens Pf- 5] 9, 10 Ammonia-lyase
4.3.1.3 Unknown Phenylalanine/histidine 77380664 98 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 11, 12 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 63 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 13, 14 Ammonia-lyase 4.3.1.3
Unknown Histidine ammonia-lyase 27359656 61 [Vibrio vulnificus
CMCP6] 15, 16 Ammonia-lyase 4.3.1.3 Unknown Histidine ammonia-lyase
27359656 61 [Vibrio vulnificus CMCP6] 17, 18 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 95 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 19, 20 Ammonia-lyase 4.3.1.3
Unknown putative histidine ammonia- 46915212 61 lyase protein
[Photobacterium profundum SS9] 21, 22 Ammonia-lyase 4.3.1.3 Unknown
putative histidine ammonia- 28805876 63 lyase protein [Vibrio
parahaemolyticus RIMD 2210633] 23, 24 Ammonia-lyase 4.3.1.3 Unknown
putative histidine ammonia- 84388067 62 lyase protein [Vibrio
splendidus 12B01] gi|84377134|gb|EAP94004.1| putative histidine
ammonia- lyase protein [Vibrio splendidus 12B01] 25, 26
Ammonia-lyase 4.3.1.3 Unknown putative histidine ammonia- 46915212
61 lyase protein [Photobacterium profundum SS9] 27, 28
Ammonia-lyase 4.3.1.3 Unknown Phenylalanine/histidine 77380664 87
ammonia-lyase [Pseudomonas fluorescens PfO-1] 29, 30 Ammonia-lyase
4.3.1.3 Unknown Phenylalanine/histidine 77380664 65 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 31, 32 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 65 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 33, 34 Ammonia-lyase 4.3.1.3
Unknown PROBABLE HISTIDINE 17430834 58 AMMONIA-LYASE PROTEIN
[Ralstonia solanacearum] 35, 36 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 37, 38 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 39, 40 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 63 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 41, 42 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 43, 44 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 63 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 45, 46 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 63 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 47, 48 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 49, 50 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 51, 52 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 63 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 53, 54 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 55, 56 Ammonia-lyase 4.3.1.3 Unknown putative
histidine ammonia- 84388067 61 lyase protein [Vibrio splendidus
12B01] gi|84377134|gb|EAP94004.1| putative histidine ammonia- lyase
protein [Vibrio splendidus 12B01] 57, 58 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 63 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 59, 60 Ammonia-lyase 4.3.1.3
Unknown Histidine ammonia-lyase 68545214 60 [Shewanella amazonensis
SB2B] gi|68517082|gb|EAN40792.1| Histidine ammonia-lyase
[Shewanella amazonensis SB2B] 61, 62 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 62 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 63, 64 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 61 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 65, 66 Ammonia-lyase 4.3.1.3 Unknown Histidine
ammonia-lyase 56178255 61 [Idiomarina Ioihiensis L2TR] 67, 68
Ammonia-lyase 4.3.1.3 Unknown Phenylalanine/histidine 77380664 62
ammonia-lyase [Pseudomonas fluorescens PfO-1] 69, 70 Ammonia-lyase
4.3.1.3 Unknown Phenylalanine/histidine 77380664 61 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 71, 72 Ammonia-lyase 4.3.1.3
Unknown Phenylalanine/histidine 77380664 62 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 73, 74 Ammonia-lyase 4.3.1.3
Unknown Histidine ammonia-lyase 27359656 60 [Vibrio vulnificus
CMCP6] 75, 76 Ammonia-lyase 4.3.1.3 Unknown Phenylalanine/histidine
77380664 61 ammonia-lyase [Pseudomonas fluorescens PfO-1] 77, 78
Ammonia-lyase 4.3.1.3 Unknown Histidine ammonia-lyase 56178255 60
[Idiomarina Ioihiensis L2TR] 79, 80 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 61 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 81, 82 Ammonia-lyase 4.3.1.3 Unknown Histidine
ammonia-lyase 27359656 60 [Vibrio vulnificus CMCP6] 83, 84
Ammonia-lyase 4.3.1.3 Unknown Histidine ammonia-lyase 56178255 61
[Idiomarina Ioihiensis L2TR] 85, 86 Ammonia-lyase 4.3.1.3 Unknown
Phenylalanine/histidine 77380664 98 ammonia-lyase [Pseudomonas
fluorescens PfO-1] 87, 88 Ammonia-lyase 4.3.1.3 Unknown Histidine
ammonia-lyase 27359656 62 [Vibrio vulnificus CMCP6] 89, 90
Ammonia-lyase 4.3.1.3 Unknown Phenylalanine/histidine 77380664 62
ammonia-lyase [Pseudomonas fluorescens PfO-1] 91, 92 Ammonia-lyase
4.3.1.3 Unknown Phenylalanine/histidine 77380664 61 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 93, 94 Ammonia-lyase 4.3.1.3
Unknown Histidine ammonia-lyase 56178255 61 [Idiomarina Ioihiensis
L2TR] 95, 96 Ammonia-lyase 4.3.1.3 Unknown Histidine ammonia-lyase
56178255 60 [Idiomarina Ioihiensis L2TR] 97, 98 Ammonia-lyase
Unknown Phenylalanine/histidine 77380664 63 ammonia-lyase
[Pseudomonas fluorescens PfO-1] 99, 100 Ammonia-lyase Unknown
Histidine ammonia-lyase 56178255 61 [Idiomarina Ioihiensis L2TR]
101, 102 Ammonia-lyase 4.3.1.3 Unknown Histidine ammonia-lyase
27359656 61 [Vibrio vulnificus CMCP6]
[0458] Polypeptides of the invention can also be shorter than the
full length of exemplary polypeptides. In alternative aspects, the
invention provides polypeptides (peptides, fragments) ranging in
size between about 5 and the full length of a polypeptide, e.g., an
enzyme, such as an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzyme; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more
residues, e.g., contiguous residues of an exemplary ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme of the invention. Peptides of the
invention (e.g., a subsequence of an exemplary polypeptide of the
invention) can be useful as, e.g., labeling probes, antigens,
toleragens, motifs, ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
active sites (e.g., "catalytic domains"), signal sequences and/or
prepro domains. By a "polypeptide having a lyase activity" is meant
a polypeptide that either by itself, or in association with one or
more additional polypeptides (having the same or a different
sequence), is a protein with the enzymatic activity of a lyase.
[0459] In one aspect, "fragments" as used herein are a portion of a
naturally occurring protein which can exist in at least two
different conformations. Fragments can have the same or
substantially the same amino acid sequence as the naturally
occurring protein. Fragments which have different three dimensional
structures as the naturally occurring protein are also included. An
example of this, is a "pro-form" molecule, such as a low activity
proprotein that can be modified by cleavage to produce a mature
enzyme with significantly higher activity.
[0460] "Amino acid" or "amino acid sequence" as used herein refer
to an oligopeptide, peptide, polypeptide, or protein sequence, or
to a fragment, portion, or subunit of any of these and to naturally
occurring or synthetic molecules. "Amino acid" or "amino acid
sequence" include an oligopeptide, peptide, polypeptide, or protein
sequence, or to a fragment, portion, or subunit of any of these,
and to naturally occurring or synthetic molecules. The term
"polypeptide" as used herein, refers to amino acids joined to each
other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres and may contain modified amino acids other than the 20
gene-encoded amino acids. The polypeptides may be modified by
either natural processes, such as post-translational processing, or
by chemical modification techniques which are well known in the
art. Modifications can occur anywhere in the polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. It will be appreciated that the same type of
modification may be present in the same or varying degrees at
several sites in a given polypeptide. Also a given polypeptide may
have many types of modifications. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of a phosphatidylinositol, cross-linking cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristolyation,
oxidation, pegylation, glucan hydrolase processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation and transfer-RNA mediated addition of amino acids to
protein such as arginylation. (See Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W.H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983)). The peptides and polypeptides of the
invention also include all "mimetic" and "peptidomimetic" forms, as
described in further detail, below.
[0461] As used herein, the term "isolated" means that the material
is removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting materials
in the natural system, is isolated. Such polynucleotides could be
part of a vector and/or such polynucleotides or polypeptides could
be part of a composition and still be isolated in that such vector
or composition is not part of its natural environment. As used
herein, the term "purified" does not require absolute purity;
rather, it is intended as a relative definition. Individual nucleic
acids obtained from a library have been conventionally purified to
electrophoretic homogeneity. The sequences obtained from these
clones could not be obtained directly either from the library or
from total human DNA. The purified nucleic acids of the invention
have been purified from the remainder of the genomic DNA in the
organism by at least 10.sup.4-10.sup.6 fold. However, the term
"purified" also includes nucleic acids which have been purified
from the remainder of the genomic DNA or from other sequences in a
library or other environment by at least one order of magnitude,
typically two or three orders and more typically four or five
orders of magnitude.
[0462] "Recombinant" polypeptides or proteins refer to polypeptides
or proteins produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide or protein. "Synthetic" polypeptides or protein
are those prepared by chemical synthesis. Solid-phase chemical
peptide synthesis methods can also be used to synthesize the
polypeptide or fragments of the invention. Such method have been
known in the art since the early 1960's (Merrifield, R. B., J. Am.
Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,
J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,
Rockford, Ill., pp. 11-12)) and have recently been employed in
commercially available laboratory peptide design and synthesis kits
(Cambridge Research Biochemicals). Such commercially available
laboratory kits have generally utilized the teachings of H. M.
Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and
provide for synthesizing peptides upon the tips of a multitude of
"rods" or "pins" all of which are connected to a single plate.
[0463] In alternative aspects, the terms "ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase" encompass any polypeptide or enzymes
capable of catalyzing the deamination of phenylalanine or tyrosine
to trans-cinnamic acid and ammonia and/or catalyzing the
abstraction of ammonia from histidine to form urocanoic acid,
including, e.g., the exemplary polypeptides and polynucleotides of
the invention (e.g., SEQ ID NO:s 1-252).
[0464] In alternative aspects, polypeptides of the invention having
ammonia lyase activity, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase activity are members
of a genus of polypeptides sharing specific structural elements,
e.g., amino acid residues that correlate with ammonia lyase
activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase activity. These shared structural
elements can be used for the routine generation of ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase variants. These shared structural elements
of ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes of the
invention can be used as guidance for the routine generation of
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes variants within the
scope of the genus of polypeptides of the invention.
[0465] Polypeptides of the invention can be used in the synthesis
or manufacture of amino acid derivatives, including .alpha. or
.beta.-amino acid derivatives, e.g. phenylalanine, histidine or
tyrosine derivatives, wherein an .alpha. or .beta.-amino acid, e.g.
phenylalanine, histidine or tyrosine, is altered by substituting a
halogen-, methyl-, ethyl-, hydroxy-, hydroxymethyl-, nitro-, or
amino-comprising group in any or all of the 2, 3, 4, and 5
positions in the aromatic side chain of the amino acid. For
example, polypeptides of the invention can be used in the synthesis
or manufacture of ortho, meta and para isomers of phenylalanine
and/or tyrosine, e.g., ortho-, meta- or para-bromo phenylalanine;
ortho-, meta- or para-fluoro phenylalanine; ortho-, meta- or
para-iodo phenylalanine; ortho-, meta- or para-chloro
phenylalanine; ortho-, meta- or para-methyl phenylalanine; ortho-,
meta- or para-hydroxyl phenylalanine; ortho-, meta- or
para-hydroxymethyl phenylalanine; ortho-, meta- or para-ethyl
phenylalanine ortho-, meta- or para-nitro phenylalanine; ortho-,
meta- or para-amino phenylalanine; ortho- or meta-bromo tyrosine;
ortho- or meta-fluoro tyrosine; ortho- or meta-iodo tyrosine;
ortho- or meta-chloro tyrosine; ortho- or meta-methyl tyrosine;
ortho- or meta-hydroxyl tyrosine; ortho- or meta-hydroxymethyl
tyrosine; ortho- or meta-ethyl tyrosine; ortho- or meta-nitro
tyrosine; ortho- or meta-amino tyrosine, all in both L and D
enantiomers, such as L- and D-.beta.-amino acids (e.g.,
L-phenylalanine and D-phenylalanine, L- and D-histidine, L- and
D-tyrosine), as well as derivatives thereof. Polypeptides of the
invention can also be used in the synthesis or manufacture of
urocanoic acid and urocanoic acid derivatives, from histidine and
histidine derivatives.
[0466] Additionally, the crystal (three-dimensional) structure of
ammonia lyases have been analyzed, e.g., see Calabrese, et al
(2004) "Crystal structure of phenylalanine ammonia lyase: multiple
helix dipoles implicated in catalysis", Biochemistry,
43(36):11403-16; Levy, et al (2002) "Insights into enzyme evolution
revealed by the structure of methylaspartate ammonia lyase",
Structure (Camb), 10(1):105-13; Baedeker, et al (2002)
"Autocatalytic peptide cyclization during chain folding of
histidine ammonia-lyase", Structure (Camb), 10(1):61-7; Schwede, et
al (1999) "Crystal structure of histidine ammonia-lyase revealing a
novel polypeptide modification as the catalytic electrophile",
Biochemistry, 27; 38(17):5355-61; Shi, et al (1997) "The structure
of L-aspartate ammonia-lyase from Escherichia coli", Biochemistry,
36(30):9136-44., illustrating specific structural elements as
guidance for the routine generation of ammonia lyase variants.
[0467] Polypeptides and peptides of the invention can be isolated
from natural sources, be synthetic, or be recombinantly generated
polypeptides. Peptides and proteins can be recombinantly expressed
in vitro or in vivo. The peptides and polypeptides of the invention
can be made and isolated using any method known in the art.
Polypeptide and peptides of the invention can also be synthesized,
whole or in part, using chemical methods well known in the art. See
e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn
(1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,
Therapeutic Peptides and Proteins, Formulation, Processing and
Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.
For example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer.
[0468] The peptides and polypeptides of the invention can also be
glycosylated. The glycosylation can be added post-translationally
either chemically or by cellular biosynthetic mechanisms, wherein
the later incorporates the use of known glycosylation motifs, which
can be native to the sequence or can be added as a peptide or added
in the nucleic acid coding sequence. The glycosylation can be
O-linked or N-linked.
[0469] The peptides and polypeptides of the invention, as defined
above, include all "mimetic" and "peptidomimetic" forms. The terms
"mimetic" and "peptidomimetic" refer to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of the polypeptides of the invention.
The mimetic can be either entirely composed of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of
partly natural peptide amino acids and partly non-natural analogs
of amino acids. The mimetic can also incorporate any amount of
natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's
structure and/or activity. As with polypeptides of the invention
which are conservative variants or members of a genus of
polypeptides of the invention (e.g., having about 50% or more
sequence identity to an exemplary sequence of the invention),
routine experimentation will determine whether a mimetic is within
the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Thus, in one aspect, a
mimetic composition is within the scope of the invention if it has
an ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes activity.
[0470] Polypeptide mimetic compositions of the invention can
contain any combination of non-natural structural components. In
alternative aspect, mimetic compositions of the invention include
one or all of the following three structural groups: a) residue
linkage groups other than the natural amide bond ("peptide bond")
linkages; b) non-natural residues in place of naturally occurring
amino acid residues; or c) residues which induce secondary
structural mimicry, i.e., to induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like. For example, a polypeptide of the
invention can be characterized as a mimetic when all or some of its
residues are joined by chemical means other than natural peptide
bonds. Individual peptidomimetic residues can be joined by peptide
bonds, other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC) Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester
(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, NY).
[0471] A polypeptide of the invention can also be characterized as
a mimetic by containing all or some non-natural residues in place
of naturally occurring amino acid residues. Non-natural residues
are well described in the scientific and patent literature; a few
exemplary non-natural compositions useful as mimetics of natural
amino acid residues and guidelines are described below. Mimetics of
aromatic amino acids can be generated by replacing by, e.g., D- or
L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;
D- or L-1, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine;
D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine;
D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where
alkyl can be substituted or unsubstituted methyl, ethyl, propyl,
hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl,
or a non-acidic amino acids. Aromatic rings of a non-natural amino
acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic
rings.
[0472] Mimetics of acidic amino acids can be generated by
substitution by, e.g., non-carboxylate amino acids while
maintaining a negative charge; (phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can
also be selectively modified by reaction with carbodiimides
(R'--N--C--N--R') such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions. Mimetics of basic amino
acids can be generated by substitution with, e.g., (in addition to
lysine and arginine) the amino acids ornithine, citrulline, or
(guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where
alkyl is defined above. Nitrile derivative (e.g., containing the
CN-moiety in place of COOH) can be substituted for asparagine or
glutamine. Asparaginyl and glutaminyl residues can be deaminated to
the corresponding aspartyl or glutamyl residues. Arginine residue
mimetics can be generated by reacting arginyl with, e.g., one or
more conventional reagents, including, e.g., phenylglyoxal,
2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, in one aspect
under alkaline conditions. Tyrosine residue mimetics can be
generated by reacting tyrosyl with, e.g., aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane can be used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Cysteine residue mimetics can be
generated by reacting cysteinyl residues with, e.g.,
alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
and corresponding amines; to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteine residue mimetics can also
be generated by reacting cysteinyl residues with, e.g.,
bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic
acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl
disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate;
2-chloromercuri-4 nitrophenol; or,
chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be
generated (and amino terminal residues can be altered) by reacting
lysinyl with, e.g., succinic or other carboxylic acid anhydrides.
Lysine and other alpha-amino-containing residue mimetics can also
be generated by reaction with imidoesters, such as methyl
picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione,
and transamidase-catalyzed reactions with glyoxylate. Mimetics of
methionine can be generated by reaction with, e.g., methionine
sulfoxide. Mimetics of proline include, e.g., pipecolic acid,
thiazolidine carboxylic acid, 3- or 4-hydroxy proline,
dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
Histidine residue mimetics can be generated by reacting histidyl
with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of
proline and lysine; phosphorylation of the hydroxyl groups of seryl
or threonyl residues; methylation of the alpha-amino groups of
lysine, arginine and histidine; acetylation of the N-terminal
amine; methylation of main chain amide residues or substitution
with N-methyl amino acids; or amidation of C-terminal carboxyl
groups.
[0473] A residue, e.g., an amino acid, of a polypeptide of the
invention can also be replaced by an amino acid (or peptidomimetic
residue) of the opposite chirality. Thus, any amino acid naturally
occurring in the L-configuration (which can also be referred to as
the R or S, depending upon the structure of the chemical entity)
can be replaced with the amino acid of the same chemical structural
type or a peptidomimetic, but of the opposite chirality, referred
to as the D-amino acid, but also can be referred to as the R- or
S-form.
[0474] The invention also provides methods for modifying the
polypeptides of the invention by either natural processes, such as
post-translational processing (e.g., phosphorylation, acylation,
etc), or by chemical modification techniques, and the resulting
modified polypeptides. Modifications can occur anywhere in the
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of a phosphatidylinositol,
cross-linking cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristolyation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, and transfer-RNA mediated addition of amino acids to
protein such as arginylation. See, e.g., Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W.H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983).
[0475] Solid-phase chemical peptide synthesis methods can also be
used to synthesize the polypeptide or fragments of the invention.
Such method have been known in the art since the early 1960's
(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See
also Stewart, J. M. and Young, J. D., Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., pp.
11-12)) and have recently been employed in commercially available
laboratory peptide design and synthesis kits (Cambridge Research
Biochemicals). Such commercially available laboratory kits have
generally utilized the teachings of H. M. Geysen et al, Proc. Natl.
Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of "rods" or "pins" all of
which are connected to a single plate. When such a system is
utilized, a plate of rods or pins is inverted and inserted into a
second plate of corresponding wells or reservoirs, which contain
solutions for attaching or anchoring an appropriate amino acid to
the pin's or rods tips. By repeating such a process step, i.e.,
inverting and inserting the rod's and pin's tips into appropriate
solutions, amino acids are built into desired peptides. In
addition, a number of available FMOC peptide synthesis systems are
available. For example, assembly of a polypeptide or fragment can
be carried out on a solid support using an Applied Biosystems, Inc.
Model 431A.TM. automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques.
[0476] The polypeptides of the invention include ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes in an active or inactive form. For
example, the polypeptides of the invention include proproteins
before "maturation" or processing of prepro sequences, e.g., by a
proprotein-processing enzyme, such as a proprotein convertase to
generate an "active" mature protein. The polypeptides of the
invention include ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes
inactive for other reasons, e.g., before "activation" by a
post-translational processing event, e.g., an endo- or
exo-peptidase or proteinase action, a phosphorylation event, an
amidation, a glycosylation or a sulfation, a dimerization event,
and the like. The polypeptides of the invention include all active
forms, including active subsequences, e.g., catalytic domains or
active sites, of the enzyme.
[0477] The invention includes immobilized ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes, anti-ammonia lyase, e.g.,
anti-phenylalanine ammonia lyase, anti-tyrosine ammonia lyase
and/or anti-histidine ammonia lyase enzyme antibodies and fragments
thereof. The invention provides methods for inhibiting ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme activity, e.g., using
dominant negative mutants or anti-ammonia lyase, e.g.,
anti-phenylalanine ammonia lyase, anti-tyrosine ammonia lyase
and/or anti-histidine ammonia lyase enzyme antibodies of the
invention. The invention includes heterocomplexes, e.g., fusion
proteins, heterodimers, etc., comprising the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention.
[0478] Polypeptides of the invention can have an ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity under various conditions,
e.g., extremes in pH and/or temperature, oxidizing agents, and the
like. The invention provides methods leading to alternative ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme preparations with different
catalytic efficiencies and stabilities, e.g., towards temperature,
oxidizing agents and changing wash conditions. In one aspect,
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme variants can be
produced using techniques of site-directed mutagenesis and/or
random mutagenesis. In one aspect, directed evolution can be used
to produce a great variety of ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme variants with alternative specificities and
stability.
[0479] The proteins of the invention are also useful as research
reagents to identify ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
modulators, e.g., activators or inhibitors of ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity. Briefly, test samples
(compounds, broths, extracts, and the like) are added to ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme assays to determine their
ability to inhibit substrate cleavage Inhibitors identified in this
way can be used in industry and research to reduce or prevent
undesired proteolysis. As with ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes, inhibitors can be combined to increase the spectrum
of activity.
[0480] The enzymes of the invention are also useful as research
reagents to digest proteins or in protein sequencing. For example,
the ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes may be used to
break polypeptides into smaller fragments for sequencing using,
e.g. an automated sequencer.
[0481] The invention also provides methods of discovering new
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes using the nucleic
acids, polypeptides and antibodies of the invention. In one aspect,
phagemid libraries are screened for expression-based discovery of
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes. In another aspect,
lambda phage libraries are screened for expression-based discovery
of ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes. Screening of
the phage or phagemid libraries can allow the detection of toxic
clones; improved access to substrate; reduced need for engineering
a host, by-passing the potential for any bias resulting from mass
excision of the library; and, faster growth at low clone densities.
Screening of phage or phagemid libraries can be in liquid phase or
in solid phase. In one aspect, the invention provides screening in
liquid phase. This gives a greater flexibility in assay conditions;
additional substrate flexibility; higher sensitivity for weak
clones; and ease of automation over solid phase screening.
[0482] The invention provides screening methods using the proteins
and nucleic acids of the invention and robotic automation to enable
the execution of many thousands of biocatalytic reactions and
screening assays in a short period of time, e.g., per day, as well
as ensuring a high level of accuracy and reproducibility (see
discussion of arrays, below). As a result, a library of derivative
compounds can be produced in a matter of weeks. For further
teachings on modification of molecules, including small molecules,
see PCT/US94/09174.
[0483] In one aspect, polypeptides or fragments of the invention
may be obtained through biochemical enrichment or purification
procedures. The sequence of potentially homologous polypeptides or
fragments may be determined by ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme assays (see, e.g., Examples 1, 2 and 3, below), gel
electrophoresis and/or microsequencing. The sequence of the
prospective polypeptide or fragment of the invention can be
compared to an exemplary polypeptide of the invention, or a
fragment, e.g., comprising at least about 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 or more consecutive amino acids thereof
using any of the programs described above.
[0484] Another aspect of the invention is an assay for identifying
fragments or variants of the invention, which retain the enzymatic
function of the polypeptides of the invention. For example the
fragments or variants of said polypeptides, may be used to catalyze
biochemical reactions, which indicate that the fragment or variant
retains the enzymatic activity of a polypeptide of the
invention.
[0485] An exemplary assay for determining if fragments of variants
retain the enzymatic activity of the polypeptides of the invention
includes the steps of: contacting the polypeptide fragment or
variant with a substrate molecule under conditions which allow the
polypeptide fragment or variant to function and detecting either a
decrease in the level of substrate or an increase in the level of
the specific reaction product of the reaction between the
polypeptide and substrate.
[0486] The present invention exploits the unique catalytic
properties of enzymes. Whereas the use of biocatalysts (i.e.,
purified or crude enzymes, non-living or living cells) in chemical
transformations normally requires the identification of a
particular biocatalyst that reacts with a specific starting
compound, the present invention uses selected biocatalysts and
reaction conditions that are specific for functional groups that
are present in many starting compounds, such as small molecules.
Each biocatalyst is specific for one functional group, or several
related functional groups and can react with many starting
compounds containing this functional group.
[0487] The biocatalytic reactions produce a population of
derivatives from a single starting compound. These derivatives can
be subjected to another round of biocatalytic reactions to produce
a second population of derivative compounds. Thousands of
variations of the original small molecule or compound can be
produced with each iteration of biocatalytic derivatization.
[0488] Enzymes react at specific sites of a starting compound
without affecting the rest of the molecule, a process which is very
difficult to achieve using traditional chemical methods. This high
degree of biocatalytic specificity provides the means to identify a
single active compound within the library. The library is
characterized by the series of biocatalytic reactions used to
produce it, a so-called "biosynthetic history". Screening the
library for biological activities and tracing the biosynthetic
history identifies the specific reaction sequence producing the
active compound. The reaction sequence is repeated and the
structure of the synthesized compound determined. This mode of
identification, unlike other synthesis and screening approaches,
does not require immobilization technologies and compounds can be
synthesized and tested free in solution using virtually any type of
screening assay. It is important to note, that the high degree of
specificity of enzyme reactions on functional groups allows for the
"tracking" of specific enzymatic reactions that make up the
biocatalytically produced library.
[0489] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility. As a result, a library of
derivative compounds can be produced in a matter of weeks, which
would take years to produce using current chemical methods.
[0490] In a particular aspect, the invention provides a method for
modifying small molecules, comprising contacting a polypeptide
encoded by a polynucleotide described herein or enzymatically
active fragments thereof with a small molecule to produce a
modified small molecule. A library of modified small molecules is
tested to determine if a modified small molecule is present within
the library, which exhibits a desired activity. A specific
biocatalytic reaction which produces the modified small molecule of
desired activity is identified by systematically eliminating each
of the biocatalytic reactions used to produce a portion of the
library and then testing the small molecules produced in the
portion of the library for the presence or absence of the modified
small molecule with the desired activity. The specific biocatalytic
reactions which produce the modified small molecule of desired
activity is optionally repeated. The biocatalytic reactions are
conducted with a group of biocatalysts that react with distinct
structural moieties found within the structure of a small molecule,
each biocatalyst is specific for one structural moiety or a group
of related structural moieties; and each biocatalyst reacts with
many different small molecules which contain the distinct
structural moiety.
[0491] Ammonia Lyase, e.g., Phenylalanine Ammonia Lyase, Tyrosine
Ammonia Lyase and/or Histidine Ammonia Lyase Enzyme Signal
Sequences, Prepro and Catalytic Domains
[0492] The invention provides ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme signal sequences (e.g., signal peptides (SPs)), prepro
domains and catalytic domains (CDs). The SPs, prepro domains and/or
CDs of the invention can be isolated or recombinant peptides or can
be part of a fusion protein, e.g., as a heterologous domain in a
chimeric protein. The invention provides nucleic acids encoding
these catalytic domains (CDs), prepro domains and signal sequences
(SPs, e.g., a peptide having a sequence comprising/consisting of
amino terminal residues of a polypeptide of the invention).
[0493] The invention provides isolated or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a
sequence as set forth in residues 1 to 11, 1 to 12, 1 to 13, 1 to
14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21,
1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to
28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36,
1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to
45, 1 to 46, 1 to 47, 1 to 48, 1 to 49, 1 to 50, or more, of a
polypeptide of the invention, including the exemplary polypeptides
of the invention, including all even-numbered sequences between SEQ
ID NO:2 and SEQ ID NO:102. In one aspect, the invention provides
signal sequences comprising the first 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or
more amino terminal residues of a polypeptide of the invention.
[0494] Methods for identifying "prepro" domain sequences and signal
sequences are well known in the art, see, e.g., Van de Ven (1993)
Crit. Rev. Oncog. 4(2):115-136. For example, to identify a prepro
sequence, the protein is purified from the extracellular space and
the N-terminal protein sequence is determined and compared to the
unprocessed form.
[0495] The invention includes polypeptides with or without a signal
sequence and/or a prepro sequence. The invention includes
polypeptides with heterologous signal sequences and/or prepro
sequences. The prepro sequence (including a sequence of the
invention used as a heterologous prepro domain) can be located on
the amino terminal or the carboxy terminal end of the protein. The
invention also includes isolated or recombinant signal sequences,
prepro sequences and catalytic domains (e.g., "active sites")
comprising sequences of the invention. The polypeptide comprising a
signal sequence of the invention can be an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme of the invention or another ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme or another enzyme or other
polypeptide.
[0496] The ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme signal
sequences (SPs) and/or prepro sequences of the invention can be
isolated peptides, or, sequences joined to another ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme or a non-ammonia lyase, e.g.,
non-phenylalanine ammonia lyase, non-tyrosine ammonia lyase and/or
non-histidine ammonia lyase polypeptide, e.g., as a fusion
(chimeric) protein. In one aspect, the invention provides
polypeptides comprising ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
signal sequences of the invention. In one aspect, polypeptides
comprising ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme signal
sequences SPs and/or prepro of the invention comprise sequences
heterologous to an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
of the invention (e.g., a fusion protein comprising an SP and/or
prepro of the invention and sequences from another ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme or a non-ammonia lyase, e.g.,
non-phenylalanine ammonia lyase, non-tyrosine ammonia lyase and/or
non-histidine ammonia lyase protein). In one aspect, the invention
provides ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes of the
invention with heterologous SPs and/or prepro sequences, e.g.,
sequences with a yeast signal sequence. An ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme of the invention can comprise a
heterologous SP and/or prepro in a vector, e.g., a pPIC series
vector (Invitrogen, Carlsbad, Calif.).
[0497] In one aspect, SPs and/or prepro sequences of the invention
are identified following identification of novel ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase polypeptides. The pathways by which
proteins are sorted and transported to their proper cellular
location are often referred to as protein targeting pathways. One
of the most important elements in all of these targeting systems is
a short amino acid sequence at the amino terminus of a newly
synthesized polypeptide called the signal sequence. This signal
sequence directs a protein to its appropriate location in the cell
and is removed during transport or when the protein reaches its
final destination. Most lysosomal, membrane, or secreted proteins
have an amino-terminal signal sequence that marks them for
translocation into the lumen of the endoplasmic reticulum. The
signal sequences can vary in length from about 10 to 65, or more,
amino acid residues. Various methods of recognition of signal
sequences are known to those of skill in the art. For example, in
one aspect, novel ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme signal
peptides are identified by a method referred to as SignalP. SignalP
uses a combined neural network which recognizes both signal
peptides and their cleavage sites. (Nielsen (1997) "Identification
of prokaryotic and eukaryotic signal peptides and prediction of
their cleavage sites." Protein Engineering 10:1-6.
[0498] It should be understood that in some aspects ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention may not have SPs
and/or prepro sequences, or "domains." In one aspect, the invention
provides the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes of
the invention lacking all or part of an SP and/or a prepro domain.
In one aspect, the invention provides a nucleic acid sequence
encoding a signal sequence (SP) and/or prepro from one ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme operably linked to a nucleic
acid sequence of a different ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme or, optionally, a signal sequence (SPs) and/or prepro
domain from a non-ammonia lyase, e.g., non-phenylalanine ammonia
lyase, non-tyrosine ammonia lyase and/or non-histidine ammonia
lyase protein may be desired.
[0499] The invention also provides isolated or recombinant
polypeptides comprising signal sequences (SPs), prepro domain
and/or catalytic domains (CDs) of the invention and heterologous
sequences. The heterologous sequences are sequences not naturally
associated (e.g., to a enzyme) with an SP, prepro domain and/or CD.
The sequence to which the SP, prepro domain and/or CD are not
naturally associated can be on the SP's, prepro domain and/or CD's
amino terminal end, carboxy terminal end, and/or on both ends of
the SP and/or CD. In one aspect, the invention provides an isolated
or recombinant polypeptide comprising (or consisting of) a
polypeptide comprising a signal sequence (SP), prepro domain and/or
catalytic domain (CD) of the invention with the proviso that it is
not associated with any sequence to which it is naturally
associated (e.g., an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
sequence). Similarly in one aspect, the invention provides isolated
or recombinant nucleic acids encoding these polypeptides. Thus, in
one aspect, the isolated or recombinant nucleic acid of the
invention comprises coding sequence for a signal sequence (SP),
prepro domain and/or catalytic domain (CD) of the invention and a
heterologous sequence (i.e., a sequence not naturally associated
with the a signal sequence (SP), prepro domain and/or catalytic
domain (CD) of the invention). The heterologous sequence can be on
the 3' terminal end, 5' terminal end, and/or on both ends of the
SP, prepro domain and/or CD coding sequence.
[0500] Hybrid (Chimeric) Ammonia Lyase, e.g., Phenylalanine Ammonia
Lyase, Tyrosine Ammonia Lyase and/or Histidine Ammonia Lyase
Enzymes and Peptide Libraries
[0501] In one aspect, the invention provides hybrid ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes and fusion proteins, including
peptide libraries, comprising sequences of the invention. The
peptide libraries of the invention can be used to isolate peptide
modulators (e.g., activators or inhibitors) of targets, such as
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzyme substrates, receptors,
enzymes. The peptide libraries of the invention can be used to
identify formal binding partners of targets, such as ligands, e.g.,
cytokines, hormones and the like. In one aspect, the invention
provides chimeric proteins comprising a signal sequence (SP),
prepro domain and/or catalytic domain (CD) of the invention or a
combination thereof and a heterologous sequence (see above).
[0502] In one aspect, the fusion proteins of the invention (e.g.,
the peptide moiety) are conformationally stabilized (relative to
linear peptides) to allow a higher binding affinity for targets.
The invention provides fusions of ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention and other
peptides, including known and random peptides. They can be fused in
such a manner that the structure of the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes is not significantly perturbed and
the peptide is metabolically or structurally conformationally
stabilized. This allows the creation of a peptide library that is
easily monitored both for its presence within cells and its
quantity.
[0503] Amino acid sequence variants of the invention can be
characterized by a predetermined nature of the variation, a feature
that sets them apart from a naturally occurring form, e.g., an
allelic or interspecies variation of an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme sequence. In one aspect, the
variants of the invention exhibit the same qualitative biological
activity as the naturally occurring analogue. Alternatively, the
variants can be selected for having modified characteristics. In
one aspect, while the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme variants screened for the optimal
combination of desired activity. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, as discussed herein for example, M13 primer mutagenesis
and PCR mutagenesis. Screening of the mutants can be done using,
e.g., assays of glucan hydrolysis. In alternative aspects, amino
acid substitutions can be single residues; insertions can be on the
order of from about 1 to 20 amino acids, although considerably
larger insertions can be done. Deletions can range from about 1 to
about 20, 30, 40, 50, 60, 70 residues or more. To obtain a final
derivative with the optimal properties, substitutions, deletions,
insertions or any combination thereof may be used. Generally, these
changes are done on a few amino acids to minimize the alteration of
the molecule. However, larger changes may be tolerated in certain
circumstances.
[0504] The invention provides ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes where the structure of the polypeptide backbone, the
secondary or the tertiary structure, e.g., an alpha-helical or
beta-sheet structure, has been modified. In one aspect, the charge
or hydrophobicity has been modified. In one aspect, the bulk of a
side chain has been modified. Substantial changes in function or
immunological identity are made by selecting substitutions that are
less conservative. For example, substitutions can be made which
more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example a alpha-helical
or a beta-sheet structure; a charge or a hydrophobic site of the
molecule, which can be at an active site; or a side chain. The
invention provides substitutions in polypeptide of the invention
where (a) a hydrophilic residues, e.g. seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine. The variants can exhibit the same qualitative
biological activity (i.e., an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity) although variants can be selected to modify
the characteristics of the ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes as needed.
[0505] In one aspect, ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzymes of the invention comprise epitopes or purification tags,
signal sequences or other fusion sequences, etc. In one aspect, the
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes of the invention can
be fused to a random peptide to form a fusion polypeptide. By
"fused" or "operably linked" herein is meant that the random
peptide and the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme are
linked together, in such a manner as to minimize the disruption to
the stability of the ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
structure, e.g., it retains ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity. The fusion polypeptide (or fusion
polynucleotide encoding the fusion polypeptide) can comprise
further components as well, including multiple peptides at multiple
loops.
[0506] In one aspect, the peptides and nucleic acids encoding them
are randomized, either fully randomized or they are biased in their
randomization, e.g. in nucleotide/residue frequency generally or
per position. "Randomized" means that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. In one aspect, the nucleic acids which give rise to
the peptides can be chemically synthesized, and thus may
incorporate any nucleotide at any position. Thus, when the nucleic
acids are expressed to form peptides, any amino acid residue may be
incorporated at any position. The synthetic process can be designed
to generate randomized nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the nucleic
acid, thus forming a library of randomized nucleic acids. The
library can provide a sufficiently structurally diverse population
of randomized expression products to affect a probabilistically
sufficient range of cellular responses to provide one or more cells
exhibiting a desired response. Thus, the invention provides an
interaction library large enough so that at least one of its
members will have a structure that gives it affinity for some
molecule, protein, or other factor.
[0507] In one aspect, an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
of the invention is a multidomain enzyme that comprises a signal
peptide, a carbohydrate binding module, an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme catalytic domain, a linker and/or
another catalytic domain.
[0508] The invention provides a means for generating chimeric
polypeptides which may encode biologically active hybrid
polypeptides (e.g., hybrid ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes). In one aspect, the original polynucleotides encode
biologically active polypeptides. The method of the invention
produces new hybrid polypeptides by utilizing cellular processes
which integrate the sequence of the original polynucleotides such
that the resulting hybrid polynucleotide encodes a polypeptide
demonstrating activities derived from the original biologically
active polypeptides. For example, the original polynucleotides may
encode a particular enzyme from different microorganisms. An enzyme
encoded by a first polynucleotide from one organism or variant may,
for example, function effectively under a particular environmental
condition, e.g. high salinity. An enzyme encoded by a second
polynucleotide from a different organism or variant may function
effectively under a different environmental condition, such as
extremely high temperatures. A hybrid polynucleotide containing
sequences from the first and second original polynucleotides may
encode an enzyme which exhibits characteristics of both enzymes
encoded by the original polynucleotides. Thus, the enzyme encoded
by the hybrid polynucleotide may function effectively under
environmental conditions shared by each of the enzymes encoded by
the first and second polynucleotides, e.g., high salinity and
extreme temperatures.
[0509] A hybrid polypeptide resulting from the method of the
invention may exhibit specialized enzyme activity not displayed in
the original enzymes. For example, following recombination and/or
reductive reassortment of polynucleotides encoding ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes, the resulting hybrid polypeptide
encoded by a hybrid polynucleotide can be screened for specialized
non-ammonia lyase, e.g., non-phenylalanine ammonia lyase,
non-tyrosine ammonia lyase and/or non-histidine ammonia lyase
enzyme activities, e.g., hydrolase, peptidase, phosphorylase, etc.,
activities, obtained from each of the original enzymes. Thus, for
example, the hybrid polypeptide may be screened to ascertain those
chemical functionalities which distinguish the hybrid polypeptide
from the original parent polypeptides, such as the temperature, pH
or salt concentration at which the hybrid polypeptide
functions.
[0510] In one aspect, the invention relates to a method for
producing a biologically active hybrid polypeptide and screening
such a polypeptide for enhanced activity by: [0511] 1) introducing
at least a first polynucleotide in operable linkage and a second
polynucleotide in operable linkage, the at least first
polynucleotide and second polynucleotide sharing at least one
region of partial sequence homology, into a suitable host cell;
[0512] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage; [0513] 3) expressing a hybrid polypeptide encoded
by the hybrid polynucleotide; [0514] 4) screening the hybrid
polypeptide under conditions which promote identification of
enhanced biological activity; and [0515] 5) isolating the a
polynucleotide encoding the hybrid polypeptide. Isolating and
Discovering Ammonia Lyase, e.g., Phenylalanine Ammonia Lyase,
Tyrosine Ammonia Lyase and/or Histidine Ammonia Lyase Enzymes
[0516] The invention provides methods for isolating and discovering
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes and the nucleic acids
that encode them. Polynucleotides or enzymes may be isolated from
individual organisms ("isolates"), collections of organisms that
have been grown in defined media ("enrichment cultures"), or,
uncultivated organisms ("environmental samples"). The organisms can
be isolated by, e.g., in vivo biopanning (see discussion, below).
The use of a culture-independent approach to derive polynucleotides
encoding novel bioactivities from environmental samples is most
preferable since it allows one to access untapped resources of
biodiversity. Polynucleotides or enzymes also can be isolated from
any one of numerous organisms, e.g. bacteria. In addition to whole
cells, polynucleotides or enzymes also can be isolated from crude
enzyme preparations derived from cultures of these organisms, e.g.,
bacteria.
[0517] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample which may
be under-represented by several orders of magnitude compared to the
dominant species.
[0518] For example, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. Polynucleotides encoding activities of
interest are isolated from such libraries and introduced into a
host cell. The host cell is grown under conditions which promote
recombination and/or reductive reassortment creating potentially
active biomolecules with novel or enhanced activities.
[0519] In vivo biopanning may be performed utilizing a FACS-based
and non-optical (e.g., magnetic) based machines. Complex gene
libraries are constructed with vectors which contain elements which
stabilize transcribed RNA. For example, the inclusion of sequences
which result in secondary structures such as hairpins which are
designed to flank the transcribed regions of the RNA would serve to
enhance their stability, thus increasing their half life within the
cell. The probe molecules used in the biopanning process consist of
oligonucleotides labeled with reporter molecules that only
fluoresce upon binding of the probe to a target molecule. These
probes are introduced into the recombinant cells from the library
using one of several transformation methods. The probe molecules
bind to the transcribed target mRNA resulting in DNA/RNA
heteroduplex molecules. Binding of the probe to a target will yield
a fluorescent signal which is detected and sorted by the FACS
machine during the screening process.
[0520] Additionally, subcloning may be performed to further isolate
sequences of interest. In subcloning, a portion of DNA is
amplified, digested, generally by restriction enzymes, to cut out
the desired sequence, the desired sequence is ligated into a
recipient vector and is amplified. At each step in subcloning, the
portion is examined for the activity of interest, in order to
ensure that DNA that encodes the structural protein has not been
excluded. The insert may be purified at any step of the subcloning,
for example, by gel electrophoresis prior to ligation into a vector
or where cells containing the recipient vector and cells not
containing the recipient vector are placed on selective media
containing, for example, an antibiotic, which will kill the cells
not containing the recipient vector. Specific methods of subcloning
cDNA inserts into vectors are well-known in the art (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory Press (1989)). In another aspect, the enzymes of
the invention are subclones. Such subclones may differ from the
parent clone by, for example, length, a mutation, a tag or a
label.
[0521] In one aspect, the signal sequences of the invention are
identified following identification of novel ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase polypeptides. The pathways by which
proteins are sorted and transported to their proper cellular
location are often referred to as protein targeting pathways. One
of the most important elements in all of these targeting systems is
a short amino acid sequence at the amino terminus of a newly
synthesized polypeptide called the signal sequence. This signal
sequence directs a protein to its appropriate location in the cell
and is removed during transport or when the protein reaches its
final destination. Most lysosomal, membrane, or secreted proteins
have an amino-terminal signal sequence that marks them for
translocation into the lumen of the endoplasmic reticulum. More
than 100 signal sequences for proteins in this group have been
determined. The sequences vary in length from 13 to 36 amino acid
residues. Various methods of recognition of signal sequences are
known to those of skill in the art. In one aspect, the peptides are
identified by a method referred to as SignalP. SignalP uses a
combined neural network which recognizes both signal peptides and
their cleavage sites. See, e.g., Nielsen (1997) "Identification of
prokaryotic and eukaryotic signal peptides and prediction of their
cleavage sites." Protein Engineering, vol. 10, no. 1, p. 1-6. It
should be understood that some of the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention may or may not
contain signal sequences. It may be desirable to include a nucleic
acid sequence encoding a signal sequence from one ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme operably linked to a nucleic acid
sequence of a different ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
or, optionally, a signal sequence from a non-ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase protein may be desired.
[0522] The microorganisms from which the polynucleotide may be
discovered, isolated or prepared include prokaryotic
microorganisms, such as Eubacteria and Archaebacteria and lower
eukaryotic microorganisms such as fungi, some algae and protozoa.
Polynucleotides may be discovered, isolated or prepared from
environmental samples in which case the nucleic acid may be
recovered without culturing of an organism or recovered from one or
more cultured organisms. In one aspect, such microorganisms may be
extremophiles, such as hyperthermophiles, psychrophiles,
psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms can be used. Such enzymes may function at
temperatures above 100.degree. C. in terrestrial hot springs and
deep sea thermal vents, at temperatures below 0.degree. C. in
arctic waters, in the saturated salt environment of the Dead Sea,
at pH values around 0 in coal deposits and geothermal sulfur-rich
springs, or at pH values greater than 11 in sewage sludge. For
example, several esterases and lipases cloned and expressed from
extremophilic organisms show high activity throughout a wide range
of temperatures and pHs.
[0523] Polynucleotides selected and isolated as hereinabove
described are introduced into a suitable host cell. A suitable host
cell is any cell which is capable of promoting recombination and/or
reductive reassortment. The selected polynucleotides are in one
aspect already in a vector which includes appropriate control
sequences. The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or in one aspect, the host cell can be a prokaryotic cell, such as
a bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation (Davis et al., 1986).
[0524] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; and plant cells. The selection
of an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0525] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described in "SV40-transformed simian
cells support the replication of early SV40 mutants" (Gluzman,
1981) and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences and
5' flanking nontranscribed sequences. DNA sequences derived from
the SV40 splice and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
[0526] In another aspect, it is envisioned the method of the
present invention can be used to generate novel polynucleotides
encoding biochemical pathways from one or more operons or gene
clusters or portions thereof. For example, bacteria and many
eukaryotes have a coordinated mechanism for regulating genes whose
products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. Thus, a gene cluster
is a group of adjacent genes that are either identical or related,
usually as to their function. An example of a biochemical pathway
encoded by gene clusters are polyketides.
[0527] Gene cluster DNA can be isolated from different organisms
and ligated into vectors, particularly vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affects high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large DNA fragments,
such as gene clusters from mixed microbial samples. One aspect is
to use cloning vectors, referred to as "fosmids" or bacterial
artificial chromosome (BAC) vectors. These are derived from E. coli
f-factor which is able to stably integrate large segments of
genomic DNA. When integrated with DNA from a mixed uncultured
environmental sample, this makes it possible to achieve large
genomic fragments in the form of a stable "environmental DNA
library." Another type of vector for use in the present invention
is a cosmid vector. Cosmid vectors were originally designed to
clone and propagate large segments of genomic DNA. Cloning into
cosmid vectors is described in detail in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press (1989). Once ligated into an appropriate vector,
two or more vectors containing different polyketide synthase gene
clusters can be introduced into a suitable host cell. Regions of
partial sequence homology shared by the gene clusters will promote
processes which result in sequence reorganization resulting in a
hybrid gene cluster. The novel hybrid gene cluster can then be
screened for enhanced activities not found in the original gene
clusters.
[0528] Methods for screening for various enzyme activities are
known to those of skill in the art and are discussed throughout the
present specification, see, e.g., Examples 1, 2 and 3, below. Such
methods may be employed when isolating the polypeptides and
polynucleotides of the invention.
[0529] In one aspect, the invention provides methods for
discovering and isolating ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase, or compounds to modify the activity of these enzymes, using
a whole cell approach. Putative clones encoding ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase from genomic DNA library can be
screened.
Screening Methodologies and "On-Line" Monitoring Devices
[0530] In practicing the methods of the invention, a variety of
apparatus and methodologies can be used to in conjunction with the
polypeptides and nucleic acids of the invention, e.g., to screen
polypeptides for ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity, to screen compounds as potential modulators, e.g.,
activators or inhibitors, of an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme activity, for antibodies that bind to a polypeptide of
the invention, for nucleic acids that hybridize to a nucleic acid
of the invention, to screen for cells expressing a polypeptide of
the invention and the like. In addition to the array formats
described in detail below for screening samples, alternative
formats can also be used to practice the methods of the invention.
Such formats include, for example, mass spectrometers,
chromatographs, e.g., high-throughput HPLC and other forms of
liquid chromatography, and smaller formats, such as 1536-well
plates, 384-well plates and so on. High throughput screening
apparatus can be adapted and used to practice the methods of the
invention, see, e.g., U.S. Patent Application No. 20020001809.
[0531] The terms "array" or "microarray" or "biochip" or "chip" as
used herein is a plurality of target elements, each target element
comprising a defined amount of one or more polypeptides (including
antibodies) or nucleic acids immobilized onto a defined area of a
substrate surface, as discussed in further detail, below.
[0532] Capillary Arrays
[0533] Nucleic acids or polypeptides of the invention can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
invention. Capillary arrays, such as the GIGAMATRIX.TM., Diversa
Corporation, San Diego, Calif.; and arrays described in, e.g., U.S.
Patent Application No. 20020080350 A1; WO 0231203 A; WO 0244336 A,
provide an alternative apparatus for holding and screening samples.
In one aspect, the capillary array includes a plurality of
capillaries formed into an array of adjacent capillaries, wherein
each capillary comprises at least one wall defining a lumen for
retaining a sample. The lumen may be cylindrical, square, hexagonal
or any other geometric shape so long as the walls form a lumen for
retention of a liquid or sample. The capillaries of the capillary
array can be held together in close proximity to form a planar
structure. The capillaries can be bound together, by being fused
(e.g., where the capillaries are made of glass), glued, bonded, or
clamped side-by-side. Additionally, the capillary array can include
interstitial material disposed between adjacent capillaries in the
array, thereby forming a solid planar device containing a plurality
of through-holes.
[0534] A capillary array can be formed of any number of individual
capillaries, for example, a range from 100 to 4,000,000
capillaries. Further, a capillary array having about 100,000 or
more individual capillaries can be formed into the standard size
and shape of a Microtiter.RTM. plate for fitment into standard
laboratory equipment. The lumens are filled manually or
automatically using either capillary action or microinjection using
a thin needle. Samples of interest may subsequently be removed from
individual capillaries for further analysis or characterization.
For example, a thin, needle-like probe is positioned in fluid
communication with a selected capillary to either add or withdraw
material from the lumen.
[0535] In a single-pot screening assay, the assay components are
mixed yielding a solution of interest, prior to insertion into the
capillary array. The lumen is filled by capillary action when at
least a portion of the array is immersed into a solution of
interest. Chemical or biological reactions and/or activity in each
capillary are monitored for detectable events. A detectable event
is often referred to as a "hit", which can usually be distinguished
from "non-hit" producing capillaries by optical detection. Thus,
capillary arrays allow for massively parallel detection of
"hits".
[0536] In a multi-pot screening assay, a polypeptide or nucleic
acid, e.g., a ligand, can be introduced into a first component,
which is introduced into at least a portion of a capillary of a
capillary array. An air bubble can then be introduced into the
capillary behind the first component. A second component can then
be introduced into the capillary, wherein the second component is
separated from the first component by the air bubble. The first and
second components can then be mixed by applying hydrostatic
pressure to both sides of the capillary array to collapse the
bubble. The capillary array is then monitored for a detectable
event resulting from reaction or non-reaction of the two
components.
[0537] In a binding screening assay, a sample of interest can be
introduced as a first liquid labeled with a detectable particle
into a capillary of a capillary array, wherein the lumen of the
capillary is coated with a binding material for binding the
detectable particle to the lumen. The first liquid may then be
removed from the capillary tube, wherein the bound detectable
particle is maintained within the capillary, and a second liquid
may be introduced into the capillary tube. The capillary is then
monitored for a detectable event resulting from reaction or
non-reaction of the particle with the second liquid.
[0538] Arrays, or "Biochips"
[0539] Nucleic acids or polypeptides of the invention can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
invention. For example, in one aspect of the invention, a monitored
parameter is transcript expression of an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme gene. One or more, or, all the
transcripts of a cell can be measured by hybridization of a sample
comprising transcripts of the cell, or, nucleic acids
representative of or complementary to transcripts of a cell, by
hybridization to immobilized nucleic acids on an array, or
"biochip." By using an "array" of nucleic acids on a microchip,
some or all of the transcripts of a cell can be simultaneously
quantified. Alternatively, arrays comprising genomic nucleic acid
can also be used to determine the genotype of a newly engineered
strain made by the methods of the invention. Polypeptide arrays"
can also be used to simultaneously quantify a plurality of
proteins. The present invention can be practiced with any known
"array," also referred to as a "microarray" or "nucleic acid array"
or "polypeptide array" or "antibody array" or "biochip," or
variation thereof. Arrays are generically a plurality of "spots" or
"target elements," each target element comprising a defined amount
of one or more biological molecules, e.g., oligonucleotides,
immobilized onto a defined area of a substrate surface for specific
binding to a sample molecule, e.g., mRNA transcripts.
[0540] In practicing the methods of the invention, any known array
and/or method of making and using arrays can be incorporated in
whole or in part, or variations thereof, as described, for example,
in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606;
6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452;
5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752;
5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752;
5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313;
WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol.
8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern
(1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes,
Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics
Supp. 21:25-32. See also published U.S. patent applications Nos.
20010018642; 20010019827; 20010016322; 20010014449; 20010014448;
20010012537; 20010008765.
Antibodies and Antibody-Based Screening Methods
[0541] The invention provides isolated or recombinant antibodies
that specifically bind to an ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme of the invention. These antibodies can be used to
isolate, identify or quantify the ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention or related
polypeptides. These antibodies can be used to isolate other
polypeptides within the scope the invention or other related
ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia
lyase and/or histidine ammonia lyase enzymes. The antibodies can be
designed to bind to an active site of an ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme. Thus, the invention provides
methods of inhibiting ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase
enzymes using the antibodies of the invention (see discussion above
regarding applications for anti-ammonia lyase, e.g.,
anti-phenylalanine ammonia lyase, anti-tyrosine ammonia lyase
and/or anti-histidine ammonia lyase enzyme compositions of the
invention).
[0542] The term "antibody" includes a peptide or polypeptide
derived from, modeled after or substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof,
capable of specifically binding an antigen or epitope, see, e.g.
Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven
Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273;
Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term
antibody includes antigen-binding portions, i.e., "antigen binding
sites," (e.g., fragments, subsequences, complementarity determining
regions (CDRs)) that retain capacity to bind antigen, including (i)
a Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Single chain
antibodies are also included by reference in the term
"antibody."
[0543] The invention provides subsequences of polypeptides of the
invention, e.g., enzymatically active or immunogenic fragments of
the enzymes of the invention, including immunogenic fragments of a
polypeptide of the invention. The invention provides compositions
comprising a polypeptide or peptide of the invention and adjuvants
or carriers and the like.
[0544] The antibodies can be used in immunoprecipitation, staining,
immunoaffinity columns, and the like. If desired, nucleic acid
sequences encoding for specific antigens can be generated by
immunization followed by isolation of polypeptide or nucleic acid,
amplification or cloning and immobilization of polypeptide onto an
array of the invention. Alternatively, the methods of the invention
can be used to modify the structure of an antibody produced by a
cell to be modified, e.g., an antibody's affinity can be increased
or decreased. Furthermore, the ability to make or modify antibodies
can be a phenotype engineered into a cell by the methods of the
invention.
[0545] Methods of immunization, producing and isolating antibodies
(polyclonal and monoclonal) are known to those of skill in the art
and described in the scientific and patent literature, see, e.g.,
Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991);
Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical
Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New
York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988)
ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications,
New York. Antibodies also can be generated in vitro, e.g., using
recombinant antibody binding site expressing phage display
libraries, in addition to the traditional in vivo methods using
animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70;
Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
[0546] The polypeptides of the invention or fragments comprising at
least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof, may also be used to generate
antibodies which bind specifically to the polypeptides or
fragments. The resulting antibodies may be used in immunoaffinity
chromatography procedures to isolate or purify the polypeptide or
to determine whether the polypeptide is present in a biological
sample. In such procedures, a protein preparation, such as an
extract, or a biological sample is contacted with an antibody
capable of specifically binding to one of the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
[0547] In immunoaffinity procedures, the antibody is attached to a
solid support, such as a bead or other column matrix. The protein
preparation is placed in contact with the antibody under conditions
in which the antibody specifically binds to one of the polypeptides
of the invention, or fragment thereof. After a wash to remove
non-specifically bound proteins, the specifically bound
polypeptides are eluted.
[0548] The ability of proteins in a biological sample to bind to
the antibody may be determined using any of a variety of procedures
familiar to those skilled in the art. For example, binding may be
determined by labeling the antibody with a detectable label such as
a fluorescent agent, an enzymatic label, or a radioisotope.
Alternatively, binding of the antibody to the sample may be
detected using a secondary antibody having such a detectable label
thereon. Particular assays include ELISA assays, sandwich assays,
radioimmunoassays and Western Blots.
[0549] Polyclonal antibodies generated against the polypeptides of
the invention, or fragments comprising at least 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof can
be obtained by direct injection of the polypeptides into an animal
or by administering the polypeptides to an animal, for example, a
nonhuman. The antibody so obtained can bind the polypeptide itself.
In this manner, even a sequence encoding only a fragment of the
polypeptide can be used to generate antibodies which may bind to
the whole native polypeptide. Such antibodies can then be used to
isolate the polypeptide from cells expressing that polypeptide.
[0550] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72, 1983) and the EBV-hybridoma technique (Cole, et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96).
[0551] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 consecutive amino acids thereof. Alternatively,
transgenic mice may be used to express humanized antibodies to
these polypeptides or fragments thereof.
[0552] Antibodies generated against the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may be
used in screening for similar polypeptides from other organisms and
samples. In such techniques, polypeptides from the organism are
contacted with the antibody and those polypeptides which
specifically bind the antibody are detected. Any of the procedures
described above may be used to detect antibody binding. One such
screening assay is described in "Methods for Measuring Cellulase
Activities", Methods in Enzymology, Vol 160, pp. 87-116.
Kits
[0553] The invention provides kits comprising the compositions,
e.g., nucleic acids, expression cassettes, vectors, cells,
transgenic seeds or plants or plant parts, polypeptides (e.g., an
ammonia lyase enzyme) and/or antibodies of the invention. The kits
also can contain instructional material teaching the methodologies
and industrial uses of the invention, as described herein.
Whole Cell Engineering and Measuring Metabolic Parameters
[0554] The methods of the invention provide whole cell evolution,
or whole cell engineering, of a cell to develop a new cell strain
having a new phenotype, e.g., a new or modified ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity, by modifying the genetic
composition of the cell. The genetic composition can be modified by
addition to the cell of a nucleic acid of the invention, e.g., a
coding sequence for an enzyme of the invention. See, e.g.,
WO0229032; WO0196551.
[0555] To detect the new phenotype, at least one metabolic
parameter of a modified cell is monitored in the cell in a "real
time" or "on-line" time frame. In one aspect, a plurality of cells,
such as a cell culture, is monitored in "real time" or "on-line."
In one aspect, a plurality of metabolic parameters is monitored in
"real time" or "on-line." Metabolic parameters can be monitored
using the ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes of
the invention.
[0556] Metabolic flux analysis (MFA) is based on a known
biochemistry framework. A linearly independent metabolic matrix is
constructed based on the law of mass conservation and on the
pseudo-steady state hypothesis (PSSH) on the intracellular
metabolites. In practicing the methods of the invention, metabolic
networks are established, including the:
[0557] identity of all pathway substrates, products and
intermediary metabolites
[0558] identity of all the chemical reactions interconverting the
pathway metabolites, the stoichiometry of the pathway
reactions,
[0559] identity of all the enzymes catalyzing the reactions, the
enzyme reaction kinetics,
[0560] the regulatory interactions between pathway components, e.g.
allosteric interactions, enzyme-enzyme interactions etc,
[0561] intracellular compartmentalization of enzymes or any other
supramolecular organization of the enzymes, and,
[0562] the presence of any concentration gradients of metabolites,
enzymes or effector molecules or diffusion barriers to their
movement.
[0563] Once the metabolic network for a given strain is built,
mathematic presentation by matrix notion can be introduced to
estimate the intracellular metabolic fluxes if the on-line
metabolome data is available. Metabolic phenotype relies on the
changes of the whole metabolic network within a cell. Metabolic
phenotype relies on the change of pathway utilization with respect
to environmental conditions, genetic regulation, developmental
state and the genotype, etc. In one aspect of the methods of the
invention, after the on-line MFA calculation, the dynamic behavior
of the cells, their phenotype and other properties are analyzed by
investigating the pathway utilization. For example, if the glucose
supply is increased and the oxygen decreased during the yeast
fermentation, the utilization of respiratory pathways will be
reduced and/or stopped, and the utilization of the fermentative
pathways will dominate. Control of physiological state of cell
cultures will become possible after the pathway analysis. The
methods of the invention can help determine how to manipulate the
fermentation by determining how to change the substrate supply,
temperature, use of inducers, etc. to control the physiological
state of cells to move along desirable direction. In practicing the
methods of the invention, the MFA results can also be compared with
transcriptome and proteome data to design experiments and protocols
for metabolic engineering or gene shuffling, etc.
[0564] In practicing the methods of the invention, any modified or
new phenotype can be conferred and detected, including new or
improved characteristics in the cell. Any aspect of metabolism or
growth can be monitored.
[0565] Monitoring Expression of an mRNA Transcript
[0566] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of an mRNA
transcript (e.g., an ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
message) or generating new (e.g., ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme) transcripts in a cell. This
increased or decreased expression can be traced by testing for the
presence of an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme of the
invention or by ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
activity assays. mRNA transcripts, or messages, also can be
detected and quantified by any method known in the art, including,
e.g., Northern blots, quantitative amplification reactions,
hybridization to arrays, and the like. Quantitative amplification
reactions include, e.g., quantitative PCR, including, e.g.,
quantitative reverse transcription polymerase chain reaction, or
RT-PCR; quantitative real time RT-PCR, or "real-time kinetic
RT-PCR" (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318;
Xia (2001) Transplantation 72:907-914).
[0567] In one aspect of the invention, the engineered phenotype is
generated by knocking out expression of a homologous gene. The
gene's coding sequence or one or more transcriptional control
elements can be knocked out, e.g., promoters or enhancers. Thus,
the expression of a transcript can be completely ablated or only
decreased.
[0568] In one aspect of the invention, the engineered phenotype
comprises increasing the expression of a homologous gene. This can
be effected by knocking out of a negative control element,
including a transcriptional regulatory element acting in cis- or
trans-, or, mutagenizing a positive control element. One or more,
or, all the transcripts of a cell can be measured by hybridization
of a sample comprising transcripts of the cell, or, nucleic acids
representative of or complementary to transcripts of a cell, by
hybridization to immobilized nucleic acids on an array.
[0569] Monitoring Expression of a Polypeptides, Peptides and Amino
Acids
[0570] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of a polypeptide
(e.g., an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme) or
generating new polypeptides in a cell. This increased or decreased
expression can be traced by determining the amount of ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme present or by ammonia lyase,
e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzyme activity assays. Polypeptides,
peptides and amino acids also can be detected and quantified by any
method known in the art, including, e.g., nuclear magnetic
resonance (NMR), spectrophotometry, radiography (protein
radiolabeling), electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, various immunological
methods, e.g. immunoprecipitation, immunodiffusion,
immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immuno-fluorescent assays, gel
electrophoresis (e.g., SDS-PAGE), staining with antibodies,
fluorescent activated cell sorter (FACS), pyrolysis mass
spectrometry, Fourier-Transform Infrared Spectrometry, Raman
spectrometry, GC-MS, and LC-Electrospray and
cap-LC-tandem-electrospray mass spectrometries, and the like. Novel
bioactivities can also be screened using methods, or variations
thereof, described in U.S. Pat. No. 6,057,103. Furthermore, as
discussed below in detail, one or more, or, all the polypeptides of
a cell can be measured using a protein array.
Pharmaceutical Compositions and Dietary Supplements
[0571] The invention provides pharmaceutical compositions, e.g.,
formulations, comprising a composition (including polypeptide,
nucleic acid, or antibody) of the invention and a pharmaceutically
acceptable excipient. The invention provides enteral and parenteral
formulations comprising compositions of the invention. For example,
the invention provides oral formulations (including or dietary
supplements) comprising a composition of the invention. The
invention provides formulations and methods for
treating/ameliorating phenylketonuria (PKU); e.g., in one aspect
the invention provides methods comprising providing a
pharmaceutical composition or dietary supplement comprising a
composition of the invention; and administering an effective amount
of the pharmaceutical composition or dietary supplement to a
subject in need thereof, thereby/ameliorating phenylketonuria
(PKU).
[0572] The invention provides methods for decreasing the levels of
phenylalanine (Phe) in a fluid or liquid, e.g., in the bloodstream
(hyperphenylalaninemia)--including bodily fluids such as cerebral
spinal fluid (CSF) and the like. The method can also be practiced
ex vivo or in vitro, or on a non-biological fluid or substance. In
this aspect, the method comprises providing a pharmaceutical
composition or dietary supplement comprising a formulation of the
invention; and administering an effective amount of the
pharmaceutical composition or dietary supplement to a subject in
need thereof.
[0573] The pharmaceutical compositions and dietary supplements used
in the methods of the invention can be administered by any means
known in the art, e.g., parenterally, topically, orally, or by
local administration, such as by aerosol or transdermally. The
compositions and dietary supplements of the invention can be
formulated as a tablet, gel, geltab, pill, implant, liquid, spray,
powder, food, feed pellet, as an injectable formulation or as an
encapsulated formulation. The pharmaceutical compositions and
dietary supplements can be formulated in any way and can be
administered in a variety of unit dosage forms depending upon the
condition or disease and the degree of illness, the general medical
condition of each patient, the resulting preferred method of
administration and the like. Details on techniques for formulation
and administration are well described in the scientific and patent
literature, see, e.g., the latest edition of Remington's
Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.
("Remington's") (e.g., Remington, The Science and Practice of
Pharmacy, 21st Edition, by University of the Sciences in
Philadelphia, Editor).
[0574] Pharmaceutical formulations and dietary supplements can be
prepared according to any method known to the art for the
manufacture of pharmaceuticals and dietary supplements. Such drugs
and dietary supplements can contain sweetening agents, flavoring
agents, coloring agents and preserving agents. A formulation (which
includes "dietary supplements") can be admixtured with nontoxic
pharmaceutically or orally acceptable excipients which are suitable
for manufacture. Formulations may comprise one or more diluents,
emulsifiers, preservatives, buffers, excipients, etc. and may be
provided in such forms as liquids, powders, emulsions, lyophilized
powders, sprays, creams, lotions, controlled release formulations,
tablets, pills, gels, on patches, in implants, etc.
[0575] Pharmaceutical formulations and dietary supplements for oral
administration can be formulated using pharmaceutically acceptable
carriers well known in the art in appropriate and suitable dosages.
Such carriers enable the pharmaceuticals and dietary supplements to
be formulated in unit dosage forms as tablets, pills, powder,
dragees, capsules, liquids, lozenges, gels, syrups, slurries,
suspensions, etc., suitable for ingestion by the patient.
Pharmaceutical preparations and dietary supplements for oral use
can be formulated as a solid excipient, optionally grinding a
resulting mixture, and processing the mixture of granules, after
adding suitable additional compounds, if desired, to obtain tablets
or dragee cores. Suitable solid excipients are carbohydrate or
protein fillers include, e.g., sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose;
and gums including arabic and tragacanth; and proteins, e.g.,
gelatin and collagen. Disintegrating or solubilizing agents may be
added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0576] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound (i.e., dosage).
Pharmaceutical preparations and dietary supplements of the
invention can also be used orally using, e.g., push-fit capsules
made of gelatin, as well as soft, sealed capsules made of gelatin
and a coating such as glycerol or sorbitol. Push-fit capsules can
contain active agents mixed with a filler or binders such as
lactose or starches, lubricants such as talc or magnesium stearate,
and, optionally, stabilizers. In soft capsules, the active agents
can be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycol with or
without stabilizers.
[0577] Aqueous suspensions can contain an active agent (e.g., a
lyase polypeptide or peptidomimetic of the invention) in admixture
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients include a suspending agent, such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethylene oxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
(e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty
acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan
mono-oleate). The aqueous suspension can also contain one or more
preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents and one or more
sweetening agents, such as sucrose, aspartame or saccharin.
Formulations can be adjusted for osmolarity.
[0578] Oil-based pharmaceuticals are particularly useful for
administration of hydrophobic formulations or active agents of the
invention. Oil-based suspensions can be formulated by suspending an
active agent (e.g., a composition of the invention) in a vegetable
oil, such as arachis oil, olive oil, sesame oil or coconut oil, or
in a mineral oil such as liquid paraffin; or a mixture of these.
See e.g., U.S. Pat. No. 5,716,928 describing using essential oils
or essential oil components for increasing bioavailability and
reducing inter- and intra-individual variability of orally
administered hydrophobic pharmaceutical compounds (see also U.S.
Pat. No. 5,858,401). The oil suspensions can contain a thickening
agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening
agents can be added to provide a palatable oral preparation, such
as glycerol, sorbitol or sucrose. These formulations and dietary
supplements can be preserved by the addition of an antioxidant such
as ascorbic acid. As an example of an injectable oil vehicle, see
Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.
[0579] The pharmaceutical formulations and dietary supplements of
the invention can also be in the form of oil-in-water emulsions.
The oily phase can be a vegetable oil or a mineral oil, described
above, or a mixture of these. Suitable emulsifying agents include
naturally-occurring gums, such as gum acacia and gum tragacanth,
naturally occurring phosphatides, such as soybean lecithin, esters
or partial esters derived from fatty acids and hexitol anhydrides,
such as sorbitan mono-oleate, and condensation products of these
partial esters with ethylene oxide, such as polyoxyethylene
sorbitan mono-oleate. The emulsion can also contain sweetening
agents and flavoring agents, as in the formulation of syrups and
elixirs. Such formulations can also contain a demulcent, a
preservative, or a coloring agent.
[0580] In the methods of the invention, the pharmaceutical
compounds and dietary supplements can also be administered by in
intranasal, intraocular and intravaginal routes including
suppositories, insufflation, powders and aerosol formulations (for
examples of steroid inhalants, see Rohatagi (1995) J. Clin.
Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.
75:107-111). Suppositories formulations can be prepared by mixing
the drug with a suitable non-irritating excipient which is solid at
ordinary temperatures but liquid at body temperatures and will
therefore melt in the body to release the drug. Such materials are
cocoa butter and polyethylene glycols.
[0581] In the methods of the invention, the pharmaceutical
compounds and dietary supplements can be delivered by
transdermally, by a topical route, formulated as applicator sticks,
solutions, suspensions, emulsions, gels, creams, ointments, pastes,
jellies, paints, powders, and aerosols.
[0582] In the methods of the invention, the pharmaceutical
compounds and dietary supplements can also be delivered as
microspheres for slow release in the body. For example,
microspheres can be administered via intradermal injection of drug
which slowly release subcutaneously; see Rao (1995) J. Biomater
Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel
formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995);
or, as microspheres for oral administration, see, e.g., Eyles
(1997) J. Pharm. Pharmacol. 49:669-674.
[0583] In the methods of the invention, the pharmaceutical
compounds can be parenterally administered, such as by intravenous
(IV) administration or administration into a body cavity or lumen
of an organ. These formulations can comprise a solution of active
agent dissolved in a pharmaceutically acceptable carrier.
Acceptable vehicles and solvents that can be employed are water and
Ringer's solution, an isotonic sodium chloride. In addition,
sterile fixed oils can be employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids
such as oleic acid can likewise be used in the preparation of
injectables. These solutions are sterile and generally free of
undesirable matter. These formulations may be sterilized by
conventional, well known sterilization techniques. The formulations
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents, e.g.,
sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate and the like. The concentration of active
agent in these formulations can vary widely, and will be selected
primarily based on fluid volumes, viscosities, body weight, and the
like, in accordance with the particular mode of administration
selected and the patient's needs. For IV administration, the
formulation can be a sterile injectable preparation, such as a
sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated using those suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation can also be a suspension in a nontoxic
parenterally-acceptable diluent or solvent, such as a solution of
1,3-butanediol. The administration can be by bolus or continuous
infusion (e.g., substantially uninterrupted introduction into a
blood vessel for a specified period of time).
[0584] The pharmaceutical compounds, formulations and dietary
supplements of the invention can be lyophilized. The invention
provides a stable lyophilized formulation comprising a composition
of the invention, which can be made by lyophilizing a solution
comprising a pharmaceutical of the invention and a bulking agent,
e.g., mannitol, trehalose, raffinose, and sucrose or mixtures
thereof. A process for preparing a stable lyophilized formulation
can include the equivalent of lyophilizing a solution about 2.5
mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a
sodium citrate buffer having a pH greater than 5.5 but less than
6.5. See, e.g., U.S. patent app. no. 20040028670.
[0585] The compositions (e.g., formulations, including dietary
supplements) of the invention can be delivered by the use of
liposomes. By using liposomes, particularly where the liposome
surface carries ligands specific for target cells, or are otherwise
preferentially directed to a specific organ, one can focus the
delivery of the active agent into target cells in vivo. See, e.g.,
U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J.
Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol.
6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.
[0586] The compositions (e.g., formulations, including dietary
supplements) of the invention can be administered for prophylactic
and/or therapeutic treatments. In therapeutic applications,
compositions are administered to a subject already suffering from a
condition, infection or disease (e.g., PKU) in an amount sufficient
to cure, alleviate or partially arrest the clinical manifestations
of the condition, infection or disease and its complications (a
"therapeutically effective amount"). In the methods of the
invention, a pharmaceutical composition is administered in an
amount sufficient to treat (e.g., ameliorate) or prevent
PKU-related conditions, diseases or symptoms, or to decrease the
amount of phenylalanine in a body fluid such as blood, serum, CSF
and the like. The amount of composition (e.g., pharmaceutical
compositions, formulations, including dietary supplements) adequate
to accomplish this is defined as a "therapeutically effective
dose." The dosage schedule and amounts effective for this use,
i.e., the "dosing regimen," will depend upon a variety of factors,
including the stage of the disease or condition, the severity of
the disease or condition, the general state of the patient's
health, the patient's physical status, age and the like. In
calculating the dosage regimen for a patient, the mode of
administration also is taken into consideration.
[0587] The dosage regimen also takes into consideration
pharmacokinetics parameters well known in the art, i.e., the active
agents' rate of absorption, bioavailability, metabolism, clearance,
and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid
Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie
51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995)
J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613;
Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest
Remington's, supra). The state of the art allows the clinician to
determine the dosage regimen for each individual patient, active
agent and disease or condition treated. Guidelines provided for
similar compositions used as pharmaceuticals can be used as
guidance to determine the dosage regiment, i.e., dose schedule and
dosage levels, administered practicing the methods of the invention
are correct and appropriate.
[0588] Single or multiple administrations of compositions (e.g.,
pharmaceutical compositions, formulations, including dietary
supplements) of the invention can be given depending on the dosage
and frequency as required and tolerated by the patient. The
compositions should provide a sufficient quantity of active agent
to effectively treat, ameliorate or prevent PKU or other
PKU-related conditions, diseases or symptoms. For example, an
exemplary pharmaceutical formulation for oral administration of a
protein of the invention is in a daily amount of between about 0.1
to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram of body
weight per day. In an alternative embodiment, dosages are from
about 1 mg to about 4 mg per kg of body weight per patient per day
are used. Lower dosages can be used, in contrast to administration
orally, into the blood stream, into a body cavity or into a lumen
of an organ. Substantially higher dosages can be used in topical or
oral administration or administering by powders, spray or
inhalation. Actual methods for preparing parenterally or
non-parenterally administrable formulations will be known or
apparent to those skilled in the art and are described in more
detail in such publications as Remington's, supra.
[0589] The compositions (e.g., pharmaceutical compositions,
formulations, including dietary supplements) of the invention can
further comprise other drugs or pharmaceuticals, e.g., compositions
for treating PKU and related symptoms or conditions. The methods of
the invention can further comprise co-administration with other
drugs or pharmaceuticals, e.g., compositions for treating septic
shock, infection, fever, pain and related symptoms or conditions.
For example, the methods and/or compositions and formulations of
the invention can be co-formulated with and/or co-administered with
antibiotics (e.g., antibacterial or bacteriostatic peptides or
proteins).
[0590] In one aspect, the polypeptide (e.g., including a
pharmaceutical composition or dietary supplement) of the invention
is chemically modified. For example, the polypeptide can be
chemically modified to produce a protected form that possesses
better specific activity, prolonged half-life, and/or reduced
immunogenicity in vivo. A polypeptide of the invention can be
modified by any means known in the art, for example, by
glycosylation, pegylation or a combination thereof.
[0591] In one aspect, the polypeptide (e.g., including a
pharmaceutical composition or dietary supplement) of the invention
is formulated by encapsulation in a liposome, or a micro- or
nano-structure, such as a nanotubule or a nano- or
microcapsule.
[0592] In one aspect, the polypeptide is formulated in a matrix
stabilized enzyme crystal. The invention also provides matrix
stabilized enzyme crystals comprising a polypeptide of the
invention for use as pharmaceutical composition or dietary
supplement, e.g., to treat or ameliorate phenylketonuria (PKU),
e.g., as described in U.S. Patent App. No. 20020182201; for
example, the formulation can be a cross-linked crystalline enzyme
and a polymer with a reactive moiety effective to adhere to the
crystal layer of the crystalline enzyme. The invention also
provides polypeptides of the invention as polymers in the form of
multimerized (e.g., multi-functional) cross-linking forms; which in
one aspect comprise a matrix stabilized enzyme crystal, e.g., a
form resistant to degradation by proteolytic enzymes; and in
alternative aspects, the cross-linking reagents comprise a
dialdehyde cross-linking reagent, such as a linear or branched
dialdehyde, or a substituted or unsubstituted glutaraldehyde
(1,5-pentanedial), malonaldehyde (1,3-propanedial), succinaldehyde
(1,4-butanedial), adipaldehyde (1,6-hexanedial), pimelaldehyde
(1,7-heptanedial), or, glutaraldehyde; in other alternative
aspects, the cross-linking reagents comprise carbodiimides,
isoxazolium derivatives, chloroformates, carbonyldiimidazole,
bis-imidoesters, bis-succinimidyl derivatives, di-isocyanates,
di-isothiocyanates, di-sylfonyl halides, bis-nitrophenyl esters,
dialdehydes, diacylazides, bis-maleimides, bis-haloacetyl
derivatives, di-alkyl halides and bis-oxiranes (e.g., as described
in U.S. Pat. No. 5,753,487).
[0593] The compositions of the invention can also be manufactured
into biocompatible matrices, e.g., sol-gels, for encapsulating a
polypeptide of the invention for use as pharmaceutical composition
or dietary supplement, e.g., to treat or ameliorate phenylketonuria
(PKU). In one aspect, compositions of the invention are
manufactured as silica-based (e.g., oxysilane) sol-gel matrices,
e.g., as described in U.S. Pat. No. 6,395,299, Pat. App. No.
20040241205. The invention also provides nano- or microcapsules
comprising a composition of the invention for use as pharmaceutical
composition or dietary supplement, e.g., to treat or ameliorate
phenylketonuria (PKU), e.g., as described in U.S. Patent App. No.
20030157181.
[0594] The pharmaceutical compositions of the invention can be
manufactured using any conventional method, e.g., mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping, melt-spinning, spray-drying, or
lyophilizing processes. Alternative pharmaceutical formulations can
be determined depending on the patient (e.g., adult or pediatric),
condition (e.g., PKU), route of administration (e.g., oral) and the
desired dosage.
[0595] Methods for determining levels of phenylalanine (Phe) in the
bloodstream (hyperphenylalaninemia)--elevated or decreased
levels--are well known in the art, and any can be used to practice
the instant invention. For example, in one aspect, blood Phe levels
are measured using an automated fluorometric or a "Guthrie test"
blood sample system; see, e.g., Kirkman (1982) Am. J. Hum. Genet.
34(5):743-752; or, Gerasimova (1989) Clinical Chemistry
35:2112-2115, modified the method of McCaman and Robins for
fluorometry of phenylalanine to a microplate assay for routine
phenylketonuria screening, and sensitivity is 15 m.mu. mol/L for a
plasma assay and 30 m.mu. mol/L for a dried blood-spot assay.
[0596] Methods for diagnosing and managing PKU patients also are
well known in the art, and any can be used to practice the instant
invention. For example, compositions (e.g., pharmaceutical
compositions, formulations, including dietary supplements) of the
invention can be administered to ameliorate hyperphenylalaninemia
blood phenylalanine levels exceeding the limits of the acceptable
upper reference range of about 2 mg/dL or 120 mmol/L. Compositions
(e.g., pharmaceutical compositions, formulations, including dietary
supplements) of the invention can be administered to ameliorate the
levels of blood phenylalanine found in patients with
phenylketonuria (PKU), including phenylalanine levels exceeding
about 20 mg/dL (1200 mmol/L), which are considered diagnostic for
PKU. The compositions (e.g., pharmaceutical compositions,
formulations, including dietary supplements) of the invention also
can be used to ameliorate nonphenylketonuric hyperphenylalaninemia,
which includes phenylalanine levels between about 2 mg/dL and about
20 mg/dL.
[0597] Compositions (e.g., pharmaceutical compositions,
formulations, including dietary supplements) of the invention can
be administered to individuals with phenylalanine levels of about 6
mg/dL (360 mmol/L) or less in patients consuming an unrestricted
diet as either an ameliorative or prophylactic treatment regimen.
Administration of compositions of the invention can be in
conjunction with dietary restrictions, e.g., indicated for patients
whose phenylalanine levels are more than about 12 mg/dL (725
mmol/L); chronic phenylalanine levels in this range reportedly
cause measurable intellectual impairment in children. Compositions
(e.g., pharmaceutical compositions, formulations, including dietary
supplements) of the invention can be administered to children with
phenylalanine levels in the intermediate range of about 6.6 to 10
mg/dL (400-600 mmol/L) or about 7-11 mg/dL (425-660 mmol/L), e.g.,
8-9 mg/dL (480-545 mmol/L), or 10 mg/dL (600 mmol/L). One study
noted that most centers in the United States recommend restricting
dietary phenylalanine when levels exceed 10 mg/dL (600 mmol/L).
Many also recommend treatment for levels exceeding 8-9 mg/dL
(480-545 mmol/L).
[0598] The British Medical Research Council Working Party on PKU
recommends dietary phenylalanine (Phe) restriction when levels
consistently exceed 6.6-10 mg/dL (400-600 mmol/L). The British
policy for dietary treatment recommends that blood Phe levels in
infants and young children be maintained between 2-6 mg/dL with
relaxation of Phe levels after childhood. Thus, in one aspect,
compositions (e.g., pharmaceutical compositions, formulations,
including dietary supplements) of the invention can be administered
to infants and young children having Phe levels over about 6 mg/dL,
for Phe level maintenance between 2-6 mg/dL.
[0599] There is a strong relationship between increasing levels of
Phe and abnormalities in the neonate. Reports have indicated that
fetuses exposed to maternal Phe levels of 3-10 mg/dL had a 24
percent incidence of microcephaly, while those exposed to levels
>20 mg/dL had a 73 percent incidence. Thus, in one aspect,
compositions (e.g., pharmaceutical compositions, formulations,
including dietary supplements) of the invention can be administered
to pregnant women having maternal Phe levels of about 3-10 mg/dL.
Similarly, congenital heart disease was not seen among offspring of
women with Phe levels <10 mg/dL and 12 percent for levels >20
mg/dL. Recent data indicates that levels of Phe above 6 mg/dL
during pregnancy are associated with significant linear decrements
in the IQ of the child through 7 years of age.
[0600] In one aspect, PAL enzymes of the invention are orally
administered; these enzymes are designed (e.g., by sequence,
covalent or noncovalent modification, or by formulation) to have
high activity and stability in gastric environment and retain
activity in an enteric environment. PAL enzymes of the invention
can be delivered via subcutaneous or via intravenous injection;
these also are designed (e.g., by sequence, covalent or noncovalent
modification, or by formulation) to have high activity levels at
physiologically relevant pHs. In one aspect, enzymes of the
invention (e.g., PAL enzymes) are designed and/or formulated as
pharmaceutical products, e.g., for PKU or related conditions, e.g.,
any form of hyperphenylalaninemia; including being designed and/or
formulated to have the appropriate activity (k.sub.cat/K.sub.M), pH
optimum and/or gastric stability. Typically, PAL characterization
is performed in two stages: I) Determination of kinetic parameters:
K.sub.cat, K.sub.M, and, II) Stability in simulated gastric
environment.
Applications--Industrial, Experimental, Food and Feed
Processing
[0601] Polypeptides (including enzymes and antibodies) and nucleic
acids of the invention can be used for a variety of industrial,
experimental, food and feed processing, nutritional and
pharmaceutical applications, e.g., for food and feed supplements,
colorants, neutraceuticals, cosmetic and pharmaceutical needs (as
discussed, above).
[0602] Polypeptides of the invention having lyase activity (e.g.,
having ammonia lyase, e.g., phenylalanine ammonia lyase (PAL),
tyrosine ammonia lyase and/or histidine ammonia lyase activity) can
catalyze the deamination of phenylalanine or tyrosine to
trans-cinnamic acid and ammonia (FIG. 5). PALs catalyze the
abstraction of ammonia from histidine to form urocanoic acid. The
enzymes of the invention can be highly selective catalysts.
[0603] The invention provides methods using enzymes of the
invention in the food and feed industries, e.g., in methods for
making food and feed products and food and feed additives. In one
aspect, the invention provides processes using enzymes of the
invention in the medical industry, e.g., to make pharmaceuticals,
neutraceuticals, food supplements and the like. In another aspect,
the enzymes of the invention can be used in the manufacture of
phenylalanine and tyrosine as well as phenylalanine and tyrosine
derivatives. In alternative aspects, the enzymes of the invention
can be used to degrade phenylalanine, tyrosine, and derivatives
thereof to manufacture cinnamic acid, para-hydroxycinnamic acid and
derivatives thereof. In yet another aspect, the enzymes of the
invention can be used in the manufacture of bulk and fine chemicals
for industrial, medicinal and agricultural use, as well as the
direct application of the enzymes themselves; for example, enzymes
(e.g., PALs) of the invention are used for enzyme substitution
therapy for the treatment/amelioration of phenylketonuria (PKU), an
inherited metabolic disease caused by a deficiency of the enzyme
phenylalanine hydroxylase.
[0604] The enzymes of the invention can catalyze reactions with
exquisite stereo-, regio- and chemo-selectivities. For example,
enzymes of the invention, including ammonia lyases, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention, can function (or
be engineered to function) in various solvents, operate at extreme
pHs (for example, high pHs and low pHs) extreme temperatures (for
example, high temperatures and low temperatures), extreme salinity
levels (for example, high salinity and low salinity) and catalyze
reactions with compounds that are structurally unrelated to their
natural, physiological substrates.
[0605] Animal Feeds and Food or Feed Additives
[0606] The invention provides methods for treating animals
(individuals) feeds and foods and food or feed additives using
enzymes of the invention, including ammonia lyases, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention, and/or the
antibodies of the invention. The invention provides animal feeds,
foods, and additives comprising ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes of the invention and/or antibodies of the invention.
The animal (individuals) can be a human, or any wild, farm or
domestic animal, or any animal.
[0607] The animal feed, or human food, additive of the invention
may be a granulated enzyme product that may readily be mixed with
feed components. Alternatively, feed or human food additives of the
invention can form a component of a pre-mix. The granulated enzyme
product of the invention may be coated or uncoated. The particle
size of the enzyme granulates can be compatible with that of feed
and pre-mix components. This provides a safe and convenient mean of
incorporating enzymes into feeds or human foods. Alternatively, the
animal feed or human food additive of the invention may be a
stabilized liquid composition. This may be an aqueous or oil-based
slurry. See, e.g., U.S. Pat. No. 6,245,546.
[0608] Ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzymes of the present
invention, in the modification of animal feed or a food, can
process the food or feed either in vitro (by modifying components
of the feed or food) or in vivo. Polypeptides of the invention can
be added to animal feed or food compositions (which include food,
e.g., dietary, supplements).
[0609] In one aspect, an enzyme of the invention is added in
combination with another enzyme, e.g., beta-galactosidases,
catalases, laccases, cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, glucose isomerases,
glycosyltransferases, lipases, phospholipases, lipooxygenases,
beta-laccases, endo-beta-1,3(4)-laccases, cutinases, peroxidases,
amylases, glucoamylases, pectinases, reductases, oxidases,
decarboxylases, phenoloxidases, ligninases, pullulanases,
arabinanases, hemicellulases, mannanases, xylolaccases, xylanases,
pectin acetyl esterases, rhamnogalacturonan acetyl esterases,
proteases, peptidases, proteinases, polygalacturonases,
rhamnogalacturonases, galactanases, pectin lyases,
transglutaminases, pectin methylesterases, cellobiohydrolases
and/or transglutaminases. These enzyme digestion products are more
digestible by the human or animal. Thus, ammonia lyase, e.g.,
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention can contribute to
the available energy of the feed or food.
[0610] In another aspect, ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme of the invention can be supplied by expressing the
enzymes directly in transgenic feed crops (as, e.g., transgenic
plants, seeds and the like), such as grains, cereals, corn, soy
bean, rape seed, lupin and the like, or human foods. As discussed
above, the invention provides transgenic plants, plant parts and
plant cells comprising a nucleic acid sequence encoding a
polypeptide of the invention. In one aspect, the nucleic acid is
expressed such that the ammonia lyase, e.g., phenylalanine ammonia
lyase, tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
of the invention is produced in recoverable quantities. The ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme can be recovered from any
plant or plant part. Alternatively, the plant or plant part
containing the recombinant polypeptide can be used as such for
improving the quality of a food or feed, e.g., improving
nutritional value, palatability, etc.
[0611] The enzyme delivery matrix of the invention is in the form
of discrete plural particles, pellets or granules. By "granules" is
meant particles that are compressed or compacted, such as by a
pelletizing, extrusion, or similar compacting to remove water from
the matrix. Such compression or compacting of the particles also
promotes intraparticle cohesion of the particles. For example, the
granules can be prepared by pelletizing the grain-based substrate
in a pellet mill. The pellets prepared thereby are ground or
crumbled to a granule size suitable for use as an adjuvant in
animal feed or human food. Since the matrix is itself approved for
use in human or animal food or feed, it can be used as a diluent
for delivery of enzymes in human or animal food or feed.
[0612] The ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
contained in the invention enzyme delivery matrix and methods is in
one aspect a thermostable ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzyme, as described herein, so as to resist inactivation of
the ammonia lyase, e.g., phenylalanine ammonia lyase, tyrosine
ammonia lyase and/or histidine ammonia lyase enzyme during
manufacture where elevated temperatures and/or steam may be
employed to prepare the palletized enzyme delivery matrix. During
digestion of feed or food containing the invention enzyme delivery
matrix, aqueous digestive fluids will cause release of the active
enzyme. Other types of thermostable enzymes and nutritional
supplements that are thermostable can also be incorporated in the
delivery matrix for release under any type of aqueous
conditions.
[0613] A coating can be applied to the invention enzyme matrix
particles for many different purposes, such as to add a flavor or
nutrition supplement to animal feed or food, to delay release of
animal feed or food supplements and enzymes in gastric conditions,
and the like. Or, the coating may be applied to achieve a
functional goal, for example, whenever it is desirable to slow
release of the enzyme from the matrix particles or to control the
conditions under which the enzyme will be released. The composition
of the coating material can be such that it is selectively broken
down by an agent to which it is susceptible (such as heat, acid or
base, enzymes or other chemicals). Alternatively, two or more
coatings susceptible to different such breakdown agents may be
consecutively applied to the matrix particles.
[0614] The invention is also directed towards a process for
preparing an enzyme-releasing matrix. In accordance with the
invention, the process comprises providing discrete plural
particles of a grain-based substrate in a particle size suitable
for use as an enzyme-releasing matrix, wherein the particles
comprise an ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzyme
encoded by an amino acid sequence of the invention.
[0615] In one aspect, the process includes compacting or
compressing the particles of enzyme-releasing matrix into granules,
which most in one aspect is accomplished by pelletizing. The mold
inhibitor and cohesiveness agent, when used, can be added at any
suitable time, and in one aspect are mixed with the grain-based
substrate in the desired proportions prior to pelletizing of the
grain-based substrate. Moisture content in the pellet mill feed in
one aspect is in the ranges set forth above with respect to the
moisture content in the finished product, and in one aspect is
about 14-15%. In one aspect, moisture is added to the feedstock in
the form of an aqueous preparation of the enzyme to bring the
feedstock to this moisture content. The temperature in the pellet
mill in one aspect is brought to about 82.degree. C. with steam.
The pellet mill may be operated under any conditions that impart
sufficient work to the feedstock to provide pellets. The pelleting
process itself is a cost-effective process for removing water from
the enzyme-containing composition.
[0616] The compositions and methods of the invention can be
practiced in conjunction with administration of prebiotics, which
are high molecular weight sugars, e.g., fructo-oligosaccharides
(FOS); galacto-oligosaccharides (GOS), GRAS (Generally Recognized
As Safe) material. These prebiotics can be metabolized by some
probiotic lactic acid bacteria (LAB). They are non-digestible by
the majority of intestinal microbes.
[0617] Treating Foods and Food Processing
[0618] The ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes of
the invention have numerous applications in food processing
industry. The invention provides methods for hydrolyzing
phenylalanine, histidine and/or tyrosine-comprising compositions,
including, e.g., a plant cell, a bacterial cell, a yeast cell, an
insect cell, or an animal cell, or any plant or plant part, or any
food or feed, a waste product and the like.
[0619] The invention provides feeds or foods comprising an ammonia
lyase, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzyme the invention, e.g., a feed,
a liquid, e.g., a beverage (such as a fruit juice or a beer), a
bread or a dough or a bread product, or a beverage precursor (e.g.,
a wort).
[0620] The food treatment processes of the invention can also
include the use of any combination of other enzymes such as
tryptophanases or tyrosine decarboxylases, laccases, catalases,
laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,
amyloglucosidases, glucose isomerases, glycosyltransferases,
lipases, phospholipases, lipooxygenases, beta-laccases,
endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, arabinanases,
hemicellulases, mannanases, xylolaccases, xylanases, pectin acetyl
esterases, rhamnogalacturonan acetyl esterases, proteases,
peptidases, proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, cellobiohydrolases and/or transglutaminases.
[0621] Waste Treatment
[0622] Enzymes of the invention, e.g., ammonia lyase, such as
phenylalanine ammonia lyase, tyrosine ammonia lyase and/or
histidine ammonia lyase enzymes of the invention can be used in a
variety of other industrial applications, e.g., in waste treatment
(in addition to, e.g., biomass conversion to fuels). For example,
in one aspect, the invention provides a solid waste digestion
process using ammonia lyase, e.g., phenylalanine ammonia lyase,
tyrosine ammonia lyase and/or histidine ammonia lyase enzymes of
the invention. The methods can comprise reducing the mass and
volume of substantially untreated solid waste. Solid waste can be
treated with an enzymatic digestive process in the presence of an
enzymatic solution (including ammonia lyase, e.g., phenylalanine
ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia
lyase enzymes of the invention) at a controlled temperature. This
results in a reaction without appreciable bacterial fermentation
from added microorganisms. The solid waste is converted into a
liquefied waste and any residual solid waste. The resulting
liquefied waste can be separated from said any residual solidified
waste. See e.g., U.S. Pat. No. 5,709,796.
[0623] In one aspect, the compositions and methods of the invention
are used for odor removal or odor reduction in animal waste
lagoons, e.g., on swine farms, and other animal waste management
systems.
[0624] The waste treatment processes of the invention can include
the use of any combination of other enzymes, including other
lyases, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase
and/or histidine ammonia lyase enzymes, and also catalases,
laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,
amyloglucosidases, glucose isomerases, glycosyltransferases,
lipases, phospholipases, lipooxygenases, beta-laccases,
endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, phytases, arabinanases,
hemicellulases, mannanases, xylolaccases, xylanases, pectin acetyl
esterases, rhamnogalacturonan acetyl esterases, proteases,
peptidases, proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, cellobiohydrolases and/or transglutaminases.
[0625] Pharmaceutical Compositions and Dietary Supplements
[0626] The invention also provides pharmaceutical compositions and
dietary supplements (e.g., dietary aids) comprising a cellulase of
the invention (e.g., enzymes having endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity). The
cellulase activity comprises endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity. In one aspect, the
pharmaceutical compositions and dietary supplements (e.g., dietary
aids) are formulated for oral ingestion, e.g., to improve the
digestibility of foods and feeds having a high cellulose or
lignocellulosic component.
[0627] Periodontal treatment compounds can comprise an enzyme of
the invention, e.g., as described in U.S. Pat. No. 6,776,979.
Compositions and methods for the treatment or prophylaxis of acidic
gut syndrome can comprise an enzyme of the invention, e.g., as
described in U.S. Pat. No. 6,468,964.
[0628] In another aspect, wound dressings, implants and the like
comprise antimicrobial (e.g., antibiotic-acting) enzymes, including
an enzyme of the invention (including, e.g., exemplary sequences of
the invention). Enzymes of the invention can also be used in
alginate dressings, antimicrobial barrier dressings, burn
dressings, compression bandages, diagnostic tools, gel dressings,
hydro-selective dressings, hydrocellular (foam) dressings,
hydrocolloid dressings, I.V dressings, incise drapes, low adherent
dressings, odor absorbing dressings, paste bandages, post operative
dressings, scar management, skin care, transparent film dressings
and/or wound closure. Enzymes of the invention can be used in wound
cleansing, wound bed preparation, to treat pressure ulcers, leg
ulcers, burns, diabetic foot ulcers, scars, IV fixation, surgical
wounds and minor wounds. Enzymes of the invention can be used to in
sterile enzymatic debriding compositions, e.g., ointments. In
various aspects, the cellulase is formulated as a tablet, gel,
pill, implant, liquid, spray, powder, food, feed pellet or as an
encapsulated formulation.
[0629] Biodefense Applications
[0630] In other aspects, cellulases of the invention (e.g., enzymes
having endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) can be used in biodefense (e.g.,
destruction of spores or bacteria comprising a lignocellulosic
material). Use of cellulases of the invention in biodefense
applications offer a significant benefit, in that they can be very
rapidly developed against any currently unknown or biological
warfare agents of the future. In addition, cellulases of the
invention can be used for decontamination of affected environments.
In aspect, the invention provides a biodefense or bio-detoxifying
agent comprising a polypeptide having a cellulase activity, wherein
the polypeptide comprises a sequence of the invention (including,
e.g., exemplary sequences of the invention), or a polypeptide
encoded by a nucleic acid of the invention (including, e.g.,
exemplary sequences of the invention), wherein optionally the
polypeptide has activity comprising endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity.
[0631] The following examples are offered to illustrate, but not to
limit the claimed invention.
EXAMPLES
Example 1
Exemplary Histidine Ammonia Lyase (HAL) Screening Assay
[0632] HAL enzyme activity can be determined as described in
Baedeker & Schulz (Eur. J. Biochem 2002, 269, 1790-1797),
wherein enzyme activity was determined as the rate of urocanate
formation, measured spectrophotometrically at 277 nm. For a
standard assay, the enzyme was preincubated at 25.degree. C. for 5
min in 2.5 mL buffer containing 0.1 M pyrophosphate (pH 9.3), 10
.mu.M ZnCl2 and 2 mM glutathione. The reaction was started by
adding 200 .mu.L of 0.5 M histidine solution and then monitored for
approximately 5 minutes.
Example 2
Exemplary Phenylalanine Ammonia Lyase (PAL) Screening Assays
[0633] In one aspect, PAL enzyme activity can be determined as
described in Rother & Retey (Eur. J. Biochem, 2002, 269,
3065-3075), by following the formation of E-cinnamate
spectrophotometrically at 30.degree. C. at 290 nm. Specifically,
the enzyme was preincubated at 30.degree. C. for 5 min in 750 mL of
0.1 M Tris/HCl pH 8.8. The reaction was performed in 1-cm quartz
cuvettes and was started by adding 250 .mu.L of a 20-mM
L-phenylalanine solution. Starting enzyme concentrations varied
between 10 and 20 .mu.g for active enzymes and between 0.3 and 0.4
mg for less active enzymes. Enzyme activity was measured every
minute for 5 minutes for more active enzymes and every 5 minutes
for 20 minutes for less active enzymes. For determination of
kinetic constants, Km and Vmax, L-phenylalanine concentrations were
varied from 0.01 to 5 mM. Kinetic constants were determined using a
double reciprocal plot. The isolated enzymes were
electrophoretically pure as verified by Coomassie Brilliant Blue
R250 staining, allowing for the measurement of turnover numbers
(kcat), using 311.313 as the molecular mass of tetrameric PAL.
[0634] In another aspect, PAL enzyme activity can be determined as
described in Kyndt et al. (FEBS Letters 2002, 512, 240-24), by
following cinnamic acid formation at 280 nm using a double beam
spectrophotometer in 10 mM Tris buffer at 35.degree. C.
Example 3
Exemplary Tyrosine Ammonia Lyase (TAL) Screening Assay
[0635] TAL enzyme activity can be determined as described in Kyndt
et al. (FEBS Letters 2002, 512, 240-24), by monitoring
p-hydroxycinnamic acid formation at 310 nm at 35.degree. C.
Example 4
Exemplary Enzyme Discovery Protocols
[0636] This example describes some exemplary protocols for cloning
and characterizing polypeptides.
[0637] Phase I: Unique Pal enzyme sequences are subcloned into a
standard expression vector and targeted for expression in E. coli.
The enzymes may be expressed with a C-terminal His tag to
facilitate purification. Functional tagged clones can be
over-expressed on 1 L shake flask scale and targeted for
purification. Any clones not active as C-terminal His tag form can
be evaluated in untagged form. Functional (untagged) clones can be
over-expressed on 1 L shake flask scale. Due to the high volume of
enzymes being evaluated, any clones that do not illustrate
functional expression can be suspended from further analysis.
Expressed, active clones can be purified at anywhere between about
50% to 85% homogeneity or more. Purified enzymes can be
characterized as follows:
[0638] I. Kinetic Characterization: pH 7.4, 37.degree. C. [0639]
Specific Activity (SA U/mg) [0640] Estimate of K.sub.cat/K.sub.M.
[0641] Enzyme with (SA) and/or K.sub.cat/K.sub.M numbers higher
than those for R. toruloides will be further characterized with
respect to K.sub.cat and K.sub.M individually.
[0642] II. Stability Characterization [0643] Performed under
simulated gastric fluid (SGF) environment.
[0644] Residual activity (% SA) will be measured after treatment to
SGF for various times.
[0645] Phase I Deliverables: (a) Kinetic characterization of
enzymes (K.sub.cat, K.sub.M). (b)
[0646] Stability characterization of enzymes in SGF. (c)
Prioritization of enzymes, partial purification, further
evaluation.
[0647] Phase I can entail, cloning, over-expression, purification
and characterization of PAL enzymes. Due to the high throughput
nature of this work any enzymes that do not express well or that
are recalcitrant to purification can be suspended from further
analysis.
[0648] Should a property of an enzyme be found to be suboptimal
during Phase I, enhancement of the required property through
evolution of the enzyme(s) can be considered. DIRECTEVOLUTION.RTM.
optimization (as described above, and e.g., in U.S. Pat. No.
6,939,689) may be performed. In some aspects, high throughput assay
for screening of the mutants is used. One of the numerous
diagnostic assays available to Phe may be applicable. Alternately,
an electrospray mass spectrometry (ESMS) assay (see, e.g., Mann, et
al. (July 2001) Annual Review of Biochemistry, Vol. 70: 437-473)
may be appropriate. Protein libraries screens for enhanced
functionality can also be used.
Example 5
Exemplary Biocatalytic Production of Para-Hydroxycinnamate
[0649] This example describes some exemplary protocols for the
biocatalytic production of para-hydroxycinnamate using enzymes of
the invention. The invention provides polypeptides having tyrosine
ammonia lyase (TAL) activity (e.g., enzymes) for the efficient
synthesis of para-hydroxycinnamate from L-tyrosine:
##STR00001##
[0650] The invention provides industrial processes for synthesizing
p-hydroxycinnamate (pHCA) from tyrosine, catalyzed by a TAL of the
invention, as shown above. In one aspect, a TAL enzyme of the
invention has a pH optimum around (about) pH 8, 9, 10, 11 or more
alkaline; and in alternative aspects has different catalytic
parameters.
[0651] In one aspect, to make the process cost-effective, the
protocol reaction is maintained pH at 7. In some situations there
is a relatively high level of product inhibition which increases at
lower pH values. Enzymes of the invention with relatively high
catalytic efficiencies of the TAL reaction at pH 7 can be used,
these enzymes are less susceptible to product inhibition. In one
aspect, enzymes of the invention capable of achieving a desired
process target of about 85% conversion at pH 7 with substrate
loading of 50-100 g/L are used. Enzymes can be characterized in
terms of their expression and specific activity. Newly discovered
or developed (e.g., engineering a sequence of the invention with
DIRECTEVOLUTION.RTM. optimization) TAL genes are cloned, expressed,
and characterized. A wide variety of bacterial genes with TAL
activity can be identified by screening environmental libraries
with nucleic acids or antibodies of the invention.
[0652] Exemplary discovery strategies: two parallel approaches to
enzyme, e.g., PAL or TAL discovery can be taken: [0653] 1.
Sequence-based discovery of new enzymes, e.g., PALs or TALs:
degenerate primers of the invention can be used to probe
environmental DNA libraries. Sequence-based discovery tools that
permit selective discovery of PALs or TALs over other ammonia
lyases are used. [0654] 2. Activity-based discovery of new TALs: a
mass spectrometry assay can be used to screen environmental DNA
library clones. In one aspect, an MS TAL assay having a throughput
of approximately 8000/week is used. In order to maximize the
effectiveness of this screen, environmental library clones can be
multiplexed.
[0655] Assay Development: As described above, two discovery
approaches can be used. For the sequence-based approach, sets of
degenerate primers are synthesized and tested. Any methods for
capturing full length genes can be used. For the activity-based
screening approach, an MS assay is integrated into an optimal
screening work-flow compatible with screening systems of the
invention. Given the limited throughput of the MS assay, clones can
be multiplexed, e.g., at 5-10 clones per well, increasing the
throughput to approximately 80,000 assays per week. A secondary MS
assay can be used to break out hits from the primary screen. Note
that results from the sequence-based approaches can be used to
cherry pick libraries for the activity screen.
[0656] Screening of DNA Libraries: Environmental DNA libraries from
multiple sources and different environments can be screened for TAL
activities. When hits are obtained the genes can be subcloned into
appropriate expression vectors and the recombinant enzymes
characterized. The activity and expression level of TALs can be
determined using assays as described herein.
[0657] Exemplary PAL Discovery Protocol: Sequence-Based
Approaches
[0658] PALs were confirmed to be active on o-Br-Phe. A predictive
bioinformatics approach was developed to distinguish PALs from HALs
at the sequence level. This has been tested and confirmed
experimentally on ammonia lyase genes. Using this approach, PALs of
the invention have been identified and demonstrated to be active on
o-Bromo Phe. Assay conditions: substrate conc.=2 mM, pH=8.5,
Temp.=30.degree. C. Express activities as units/mL of lysate;
activities need not be normalized for expression.
[0659] An exemplary activity-based discovery protocol comprises use
of an ammonia selection using .alpha.-methylPhe to screen
environmental libraries. Using this particular assay, PALs do not
appear to be active on .alpha.-methylPhe. A phenylalanine selection
screen was also implemented to screen libraries, e.g.,
environmental libraries--which yielded, inter alia, chorismate
mutases (CM), which complement a PheA mutation in the auxotroph
screening host. An adamantyl phosphonate, an inhibitor of CM
activity, also was used to determine whether can minimize
background CM activity in this selection/screening protocol. This
inhibitor was found not be suitable because of lack of potency.
[0660] In round of screening, colonies were isolated that grew on
.alpha.-methylPhe and .alpha.-methylTyr. Isolates were frozen as
glycerol stocks; then grown on carbon-based medium supplemented
with 5 mM .alpha.-methyl phenylalanine as sole nitrogen source.
LC/MS analysis of cell-free extract activities of these isolates on
.alpha.-methyl phenylalanine indicates no formation of cinnamic
acid derivatives.
[0661] In summary, using a bioinformatics-driven approach, a
sequence-based PAL discovery technology was used to probe
environmental DNA libraries for the presence of new PALs. In
summary sequence-based discovery of new PALs produced leads that
showed activity on ortho-bromo phenylalanine.
Example 6
Exemplary Phenylalanine Ammonia Lyases
[0662] This example describes the screening and characterization of
exemplary phenylalanine ammonia lyases of the invention for the
synthesis of ortho-halo phenylalanine derivatives in high yield and
high ee. In one aspect, the invention provides a selection and a
screen for use in the discovery of PALs from libraries, e.g.,
environmental DNA libraries.
[0663] High Throughput Assay Development:
[0664] Ammonia Selection: [0665] Used clone of Rhodotorula glutinis
PAL in pUC57 vector, DH5.alpha. host. [0666] Phe, 2-ChloroPhe, and
2-BromoPhe all give background growth with negative control
(host+empty vector). .alpha.-methylPhe gives no background growth.
[0667] Positive control (host+Rhodotorula PAL) not active on
.alpha.-methylPhe. [0668] New positive control (host+SEQ ID NO:104
(encoded by, e.g., SEQ ID NO:103)) is active on .alpha.-methylPhe.
[0669] Environmental libraries are screened using .alpha.-methyl
selection approach, including actinomycete PAC libraries and
streptomycete small insert libraries.
[0670] Phenylalanine Selection: [0671] Complementary to ammonia
selection and does not require substrate analog. [0672] Obtained
auxotrophic strain from ATCC. [0673] Strain is able to grow in
presence of up to 25 mM cinnamic acid and up to 50 mM ammonium ion
(pH dependent) on minimal media+phenylalanine. Therefore cinnamic
acid and ammonia not toxic at these levels. [0674] Made competent
cells and transformed with PAL vector to generate a positive
control for selection development. [0675] Proof-of-principle
experiments for Phe selection in progress using Phe auxotroph and
positive control. Investigating growth with cinnamic acid and
ammonium ion in absence of Phe. [0676] Strain development for
library-compatible host.
[0677] High Throughput Fluorogenic Assay: [0678] Developed
fluorogenic assay based on fluorescence of ortho-hydroxycinnamic
acid at high pH. [0679] SEQ ID NO:104 (encoded by, e.g., SEQ ID
NO:103) and R. glutinis PALs show no activity on
ortho-hydroxyphenylalanine.
[0680] Analytical Assays
[0681] Non-Chiral Methods [0682] LC/MS assay: [0683] Developed
one-minute LC-MS methods for the following substrate/product pairs:
[0684] Cinnamic acid and phenylalanine; [0685] 2-bromocinnamic acid
and 2-bromophenylalanine; [0686] .alpha.-methyl cinnamic acid and
.alpha.-methylphenylalanine [0687] This medium throughput assay can
be used for screening, e.g., evolution libraries [0688]
Spectrophotometric assay: implemented continuous spectroscopic
assay based on absorbance of cinnamic acid (or derivatives) at 290
nm. [0689] Tested the following substrates: L-Phenylalanine,
2-Chloro-L-phenylalanine, 2-Bromo-L-phenylalanine,
.alpha.-Methyl-DL-phenylalanine,
.alpha.-Methyl-L-phenylalanine.
[0690] Chiral Method [0691] Developed chiral HPLC method to
separate L-2-bromophenylalanine from D-2-bromophenylalanine
[0692] New PAL Discovery
[0693] Sequence-Based Discovery [0694] bacterial PAL clone SEQ ID
NO:104 (encoded by, e.g., SEQ ID NO:103) subcloned into E. coli
expression vector. [0695] PAL activity on phenylalanine confirmed.
[0696] In contrast to R. glutinis PAL, PAL SEQ ID NO:52 shown to
have activity on .alpha.-methylphenylalanine [0697] PAL SEQ ID
NO:104 also shown to have activity on 2-bromoPhe; ratio of
2-bromoPhe activity to Phe activity appears to be higher for SEQ ID
NO:104 vs R. glutinis PAL. [0698] additional bacterial PALs can be
identified by sequence homology and subcloned. [0699] fungal PALs
can be identified and cloned from cDNA: sources include Botrytis
sp., Fusarium sp. [0700] enzymes of the invention can be expressed
in fungi or bacteria, e.g., in E. coli, Cochliobolus heterotrophus,
and/or Pichia pastoris.
[0701] Activity-Based Discovery: [0702] an ammonia selection
screening process using .alpha.-methylPhe or bromoPhe under process
conditions can be used to screen environmental libraries. [0703]
Phe auxotroph-based selection can also be used to identify
PALs.
[0704] Summary: The invention provides methods and compositions for
discovering new lyases, e.g., PALs, using nucleic acids (e.g.,
probes) and polypeptides (e.g., antibodies) of the invention. In
exemplary protocols described herein, several PAL discovery
strategies were pursued in parallel; in one aspect, environmental
libraries were screened using the ammonia-based selection and
.alpha.-methylphenylalanine. In another aspect, a complementary
selection strategy based on a phenylalanine auxotroph is used. In
another aspect, a sequence-based method is used. In one exemplary
protocol, when a new putative PAL is identified, e.g., in a library
by sequence homology, it is cloned, expressed, and tested for
enzyme activity (e.g., PAL activity).
A number of aspects of the invention have been described.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of the
invention. Accordingly, other aspects are within the scope of the
following claims.
Sequence CWU 1
1
10411536DNAUnknownObtained from environmental sample 1atgacttcac
tgatttttga tcaagcgcga ctcactattg aagacgtact tgacgccgcc 60catcagcgcc
gcacttgtgt attgagtgat gcaccggatt ttgtccagcg tattgaacag
120ggcgctcgct ttctcgaccg cctgttgcaa gaagacggcg tgatttacgg
tgtaaccacc 180ggttatggcg attcctgcac cacccatatt ccgcgtgagc
tggtcgagca attaccacaa 240catctgtaca cctttcacgg ttgtggattg
ggcgcggatt tatcgccaac cgctggccgt 300gcggtgctgg tggtacgcct
tacatcgcta actcgcggtt attcaggtgt aagttatggc 360ttgttgcaac
agctcactcg cctgctgaaa gacgatgtta ttccggtgat tcctgaagaa
420ggatcggtcg gtgccagtgg cgatttaacg ccgctatcct atgtagccgc
cgtgctctgt 480ggcgagcgcg aggtttatca tcgcggtgcg cgtcgcccaa
cagccgaggt atttgcggaa 540ctcaacatta ctccgctgac gttacgcccg
aaagaaggtt tggctttgat gaacggcact 600gcagtaatga ccgcactggc
ttgcctcgcc tatgaacgca cggcctatct tgcgcaattg 660gctacgcgga
ttaccgcaat tatgacggtt gccgcccaag gcaatgcgca ccactttgat
720gaattattat ttgccgtaaa acctcacccc ggccagcagc aagtcgccgc
ttggctgcgc 780gaagatttac acgcaggcga accgccgcgc aatccaagtc
gcttacagga tcgttattcc 840ctgcgctgtg cgccccacgt gattggcgtt
gcgaccgaca gcttgccttg gtggcgccag 900tttattgaga acgaactcaa
cagcgctaat gacaatccga ttattgatgg cgaaggcgag 960cacgtactgc
acggtggcca tttttacggg ggccacatcg ctttcgtgat ggattccatg
1020aaaaacgtgg tcgccaatat cgccgaccta cttgaccgcc aactcgcgca
gttagtggac 1080accaaattca accacggttt gccggccaat ctttccggtg
cacctgaagc gcgacgtgcg 1140attaaccacg gcttcaaggc cgtgcaaatt
ggcgtttccg cctggacagc tgaagccctc 1200aaactcacca tgcccgccag
tgttttttca cgctcaaccg aatgccacaa tcaagacaaa 1260gtcagcatgg
gcaccatcgc cgcgcgtgat tgcttgcgca tattgcagct cactgaacaa
1320gttgccgccg ccgtattgtt ggctgcctgc caaggcttgg aactgcgcca
gcaaaccgga 1380acctgcgaac acagtgtcga aggcgggatt aaaactacgc
tcgaagaaat tcgcgcgcag 1440catccctttt tggtagaaga ccgcgccttt
gatcacgagc ttcgtcagtt aattacgcgc 1500attcagcaac aggggcttag
cctttattcg gcctag 15362511PRTUnknownObtained from environmental
sample 2Met Thr Ser Leu Ile Phe Asp Gln Ala Arg Leu Thr Ile Glu Asp
Val1 5 10 15Leu Asp Ala Ala His Gln Arg Arg Thr Cys Val Leu Ser Asp
Ala Pro 20 25 30Asp Phe Val Gln Arg Ile Glu Gln Gly Ala Arg Phe Leu
Asp Arg Leu 35 40 45Leu Gln Glu Asp Gly Val Ile Tyr Gly Val Thr Thr
Gly Tyr Gly Asp 50 55 60Ser Cys Thr Thr His Ile Pro Arg Glu Leu Val
Glu Gln Leu Pro Gln65 70 75 80His Leu Tyr Thr Phe His Gly Cys Gly
Leu Gly Ala Asp Leu Ser Pro 85 90 95Thr Ala Gly Arg Ala Val Leu Val
Val Arg Leu Thr Ser Leu Thr Arg 100 105 110Gly Tyr Ser Gly Val Ser
Tyr Gly Leu Leu Gln Gln Leu Thr Arg Leu 115 120 125Leu Lys Asp Asp
Val Ile Pro Val Ile Pro Glu Glu Gly Ser Val Gly 130 135 140Ala Ser
Gly Asp Leu Thr Pro Leu Ser Tyr Val Ala Ala Val Leu Cys145 150 155
160Gly Glu Arg Glu Val Tyr His Arg Gly Ala Arg Arg Pro Thr Ala Glu
165 170 175Val Phe Ala Glu Leu Asn Ile Thr Pro Leu Thr Leu Arg Pro
Lys Glu 180 185 190Gly Leu Ala Leu Met Asn Gly Thr Ala Val Met Thr
Ala Leu Ala Cys 195 200 205Leu Ala Tyr Glu Arg Thr Ala Tyr Leu Ala
Gln Leu Ala Thr Arg Ile 210 215 220Thr Ala Ile Met Thr Val Ala Ala
Gln Gly Asn Ala His His Phe Asp225 230 235 240Glu Leu Leu Phe Ala
Val Lys Pro His Pro Gly Gln Gln Gln Val Ala 245 250 255Ala Trp Leu
Arg Glu Asp Leu His Ala Gly Glu Pro Pro Arg Asn Pro 260 265 270Ser
Arg Leu Gln Asp Arg Tyr Ser Leu Arg Cys Ala Pro His Val Ile 275 280
285Gly Val Ala Thr Asp Ser Leu Pro Trp Trp Arg Gln Phe Ile Glu Asn
290 295 300Glu Leu Asn Ser Ala Asn Asp Asn Pro Ile Ile Asp Gly Glu
Gly Glu305 310 315 320His Val Leu His Gly Gly His Phe Tyr Gly Gly
His Ile Ala Phe Val 325 330 335Met Asp Ser Met Lys Asn Val Val Ala
Asn Ile Ala Asp Leu Leu Asp 340 345 350Arg Gln Leu Ala Gln Leu Val
Asp Thr Lys Phe Asn His Gly Leu Pro 355 360 365Ala Asn Leu Ser Gly
Ala Pro Glu Ala Arg Arg Ala Ile Asn His Gly 370 375 380Phe Lys Ala
Val Gln Ile Gly Val Ser Ala Trp Thr Ala Glu Ala Leu385 390 395
400Lys Leu Thr Met Pro Ala Ser Val Phe Ser Arg Ser Thr Glu Cys His
405 410 415Asn Gln Asp Lys Val Ser Met Gly Thr Ile Ala Ala Arg Asp
Cys Leu 420 425 430Arg Ile Leu Gln Leu Thr Glu Gln Val Ala Ala Ala
Val Leu Leu Ala 435 440 445Ala Cys Gln Gly Leu Glu Leu Arg Gln Gln
Thr Gly Thr Cys Glu His 450 455 460Ser Val Glu Gly Gly Ile Lys Thr
Thr Leu Glu Glu Ile Arg Ala Gln465 470 475 480His Pro Phe Leu Val
Glu Asp Arg Ala Phe Asp His Glu Leu Arg Gln 485 490 495Leu Ile Thr
Arg Ile Gln Gln Gln Gly Leu Ser Leu Tyr Ser Ala 500 505
51031545DNAUnknownObtained from environmental sample 3atgacgacgc
caacccatga gccggtaacc ttcggcgaac gccctttgcg catcgaagac 60gtactggccc
tggccaatcg tcaggcgccc gtgcagttgc agagcgacgc cgactaccgt
120gagcgcatcg ccaaaggtgc gcggttcctc gactcgctgc tggacaagga
aggcgtgatt 180tacggcgtga ccaccggtta cggcgactcc tgcgtggttg
cggtgccgct gcatcacgtc 240gaagcattgc cacagcacct ctacacgttc
catggctgcg ggctgggcaa actgctcgac 300gcccaggcta cccgcgcggt
gctggcggcg cgtttgcagt cgctgtgcca cggcgtgtcc 360ggggtgcgca
tcgaactgct ggagcgcctg catgcgtttc ttgaacacga catcctgccg
420ctgatcccgg aagagggttc ggtgggcgcc agcggcgatc tgacgccgct
gtcctacgtc 480gccgcgaccc tgtccggcga gcgtgaagtg atgttccaag
gtcagcgccg ccagtccgcc 540gatgtgcatc gtgaactggg ctggaccccg
ctggtgctgc gcccgaaaga agcgctggca 600ctgatgaacg gcaccgccgt
gatgaccggc ctcgcttgcc tggcttacgc ccgcgccgat 660tacctgctgc
aactggccac ccgcatcacc gcgttgaacg tggtggcgct gcaaggcaat
720ccggagcact tcgacgagcg cctgttcgcc gccaaaccgc atccgggtca
gatgcaggtg 780gccgcgtggc tgcgcaagga tctggcgatc gacgcgccga
ccgcgccgct gcaccgcttg 840caggatcgct attccctgcg ctgcgcaccg
catgtgcttg gcgtattggc cgacagcctg 900agctggctgc gctcgttcat
cgagaccgaa ctcaacagcg ccaacgacaa tccgatcatc 960gacgccgaag
ccgagcgcgt gctgcacggc gggcacttct atggcgggca tatcgcgttc
1020gccatggaca gcctgaaaaa cctcgtggcc aacgtcgccg acctgctcga
ccgacaactc 1080gcgctgctgg tggacgagcg ttacaaccac ggtttgccga
gcaacctgtc cggcgccagc 1140gctgatcggg cgatgctcaa ccacggcttc
aaggctgtgc agatcggtgc cagcgcctgg 1200accgccgaag cgttgaaaaa
caccatgccc gccagcgtgt tctcgcgctc taccgagtgc 1260cacaaccagg
acaaggtgag catgggcacc atcgccgccc gcgacgccat tcgtgtgctg
1320gagctgaccg aacaggtcgc cgccgccacc ttgctcgcgg ccaatcaggg
cgtgtggctg 1380cgcagccgtg atgaggacgc gcgagcgctg ccaccgtccc
tggccgccat gcacgaagag 1440ctggcccaag acttcccgcc agtgatcgaa
gaccgcgcgc tggaaggcga actgcgcctg 1500tgcctgcaac gcatcgccga
acaacactgg aggctgcatg cgtag 15454514PRTUnknownObtained from
environmental sample 4Met Thr Thr Pro Thr His Glu Pro Val Thr Phe
Gly Glu Arg Pro Leu1 5 10 15Arg Ile Glu Asp Val Leu Ala Leu Ala Asn
Arg Gln Ala Pro Val Gln 20 25 30Leu Gln Ser Asp Ala Asp Tyr Arg Glu
Arg Ile Ala Lys Gly Ala Arg 35 40 45Phe Leu Asp Ser Leu Leu Asp Lys
Glu Gly Val Ile Tyr Gly Val Thr 50 55 60Thr Gly Tyr Gly Asp Ser Cys
Val Val Ala Val Pro Leu His His Val65 70 75 80Glu Ala Leu Pro Gln
His Leu Tyr Thr Phe His Gly Cys Gly Leu Gly 85 90 95Lys Leu Leu Asp
Ala Gln Ala Thr Arg Ala Val Leu Ala Ala Arg Leu 100 105 110Gln Ser
Leu Cys His Gly Val Ser Gly Val Arg Ile Glu Leu Leu Glu 115 120
125Arg Leu His Ala Phe Leu Glu His Asp Ile Leu Pro Leu Ile Pro Glu
130 135 140Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser
Tyr Val145 150 155 160Ala Ala Thr Leu Ser Gly Glu Arg Glu Val Met
Phe Gln Gly Gln Arg 165 170 175Arg Gln Ser Ala Asp Val His Arg Glu
Leu Gly Trp Thr Pro Leu Val 180 185 190Leu Arg Pro Lys Glu Ala Leu
Ala Leu Met Asn Gly Thr Ala Val Met 195 200 205Thr Gly Leu Ala Cys
Leu Ala Tyr Ala Arg Ala Asp Tyr Leu Leu Gln 210 215 220Leu Ala Thr
Arg Ile Thr Ala Leu Asn Val Val Ala Leu Gln Gly Asn225 230 235
240Pro Glu His Phe Asp Glu Arg Leu Phe Ala Ala Lys Pro His Pro Gly
245 250 255Gln Met Gln Val Ala Ala Trp Leu Arg Lys Asp Leu Ala Ile
Asp Ala 260 265 270Pro Thr Ala Pro Leu His Arg Leu Gln Asp Arg Tyr
Ser Leu Arg Cys 275 280 285Ala Pro His Val Leu Gly Val Leu Ala Asp
Ser Leu Ser Trp Leu Arg 290 295 300Ser Phe Ile Glu Thr Glu Leu Asn
Ser Ala Asn Asp Asn Pro Ile Ile305 310 315 320Asp Ala Glu Ala Glu
Arg Val Leu His Gly Gly His Phe Tyr Gly Gly 325 330 335His Ile Ala
Phe Ala Met Asp Ser Leu Lys Asn Leu Val Ala Asn Val 340 345 350Ala
Asp Leu Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Glu Arg Tyr 355 360
365Asn His Gly Leu Pro Ser Asn Leu Ser Gly Ala Ser Ala Asp Arg Ala
370 375 380Met Leu Asn His Gly Phe Lys Ala Val Gln Ile Gly Ala Ser
Ala Trp385 390 395 400Thr Ala Glu Ala Leu Lys Asn Thr Met Pro Ala
Ser Val Phe Ser Arg 405 410 415Ser Thr Glu Cys His Asn Gln Asp Lys
Val Ser Met Gly Thr Ile Ala 420 425 430Ala Arg Asp Ala Ile Arg Val
Leu Glu Leu Thr Glu Gln Val Ala Ala 435 440 445Ala Thr Leu Leu Ala
Ala Asn Gln Gly Val Trp Leu Arg Ser Arg Asp 450 455 460Glu Asp Ala
Arg Ala Leu Pro Pro Ser Leu Ala Ala Met His Glu Glu465 470 475
480Leu Ala Gln Asp Phe Pro Pro Val Ile Glu Asp Arg Ala Leu Glu Gly
485 490 495Glu Leu Arg Leu Cys Leu Gln Arg Ile Ala Glu Gln His Trp
Arg Leu 500 505 510His Ala51557DNAUnknownObtained from
environmental sample 5atgcaacccg ctgatttaac ctccacgcca cgtaccgtcc
gtttcgacca gggcaggttg 60caaatcgaag acgtagtcga tattgcgaat ggctctgcgc
gggcggtctt gtccgatgca 120cccgaattcc gcgccgcgat agcgcgtgga
gcagacttcc tggatcgcct gttgcgtgaa 180gacgggacga tctatggcgt
gacaacaggt tatggtgatt catgcaccgt cacagtaccc 240gcagacctgg
tcgccgagct accccatcat ttatacacct atcacggttg cggactggga
300gagtacttca ctcctgcaca aacacgtgcg atcctggctg cgcggcttgc
atcgctgagc 360aagggttatt ccggcgtcag cgtcgagctg ctgcaacaaa
tcgtgcgtct gctcgaccgc 420ggtttgttgc ccgtgatccc atccgaaggt
tcggtcggcg ccagcggtga tctgactccg 480ttgtcctacc ttgctgccgt
gctgtgtggc gagcgcgaag tatggcgtga cggcacacag 540atctttgcaa
aacaagcatt ggccgaagct ggtatcacac cactccgttt gcgcccgaaa
600gaaggattgg cgatcatgaa tggcactgct gtgatgactg ctttggcctg
catcgcatat 660tcacgtgccg aatacctgac gcaattgacc actcgcatca
cggcgatggc ttccttcgca 720ctcgatggca atgcgcatca tttcaacgaa
gtgctgtttt ctgtaaagcc gcatcctggc 780atgcagaaag tagcaggttg
gttgcgccat aacttgccat gcgagtctgc gccgcgcaat 840ggaaaacgtt
tgcaggatcg ctattcgatc cgctgtgcgc cgcatgtgat tggcgtactg
900gccgacgcgc tgccgtggtt taaacaatca attgaaaacg aactcaatag
cgccaacgac 960aacccgatca tcgatgctga aggtgaacat gtgctgcatg
gcggccattt ctatggcggg 1020catattgcgt ttgccatgga cggtatgaag
aatgcagtcg ccaatcttgc cgatttgctg 1080gatcgccaga tggcgttgct
ggtcgatagc cgttacaaca atggtttgcc ggccaatttg 1140tccggcatgg
aaggaccgcg cgctgcgatc aaccacggac tcaaggcttt gcaaatcagc
1200gtatctgcgt ggaccgcaga agcgctcaag ttgacaatgc cggcatcggt
attttcccgt 1260tcaaccgaat gccataacca ggacaaggtc agcatgggca
cgatcgcagc gcgtgactgt 1320ttgcgtgtgc tggaattgac cgaacaagtc
gtggcggcac tcttgattac cgtgcagcag 1380ggtgtttggc tgcgtcgaaa
attgaattcg tcagtcacac ctgagccttc agtggaagcg 1440atgttgggtg
cattaagtgc ggacgttcgg atgattgaag aagaccgccg attggatccg
1500gacttgaaat tgctgttgga acgtatccgc gtccgttcat ggaagttgta tgactaa
15576518PRTUnknownObtained from environmental sample 6Met Gln Pro
Ala Asp Leu Thr Ser Thr Pro Arg Thr Val Arg Phe Asp1 5 10 15Gln Gly
Arg Leu Gln Ile Glu Asp Val Val Asp Ile Ala Asn Gly Ser 20 25 30Ala
Arg Ala Val Leu Ser Asp Ala Pro Glu Phe Arg Ala Ala Ile Ala 35 40
45Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Asp Gly Thr Ile
50 55 60Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val Thr Val
Pro65 70 75 80Ala Asp Leu Val Ala Glu Leu Pro His His Leu Tyr Thr
Tyr His Gly 85 90 95Cys Gly Leu Gly Glu Tyr Phe Thr Pro Ala Gln Thr
Arg Ala Ile Leu 100 105 110Ala Ala Arg Leu Ala Ser Leu Ser Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Glu Leu Leu Gln Gln Ile Val Arg
Leu Leu Asp Arg Gly Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Val Leu Cys Gly Glu Arg Glu Val Trp Arg 165 170 175Asp
Gly Thr Gln Ile Phe Ala Lys Gln Ala Leu Ala Glu Ala Gly Ile 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ala Val Met Thr Ala Leu Ala Cys Ile Ala Tyr Ser Arg
Ala Glu 210 215 220Tyr Leu Thr Gln Leu Thr Thr Arg Ile Thr Ala Met
Ala Ser Phe Ala225 230 235 240Leu Asp Gly Asn Ala His His Phe Asn
Glu Val Leu Phe Ser Val Lys 245 250 255Pro His Pro Gly Met Gln Lys
Val Ala Gly Trp Leu Arg His Asn Leu 260 265 270Pro Cys Glu Ser Ala
Pro Arg Asn Gly Lys Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Phe Lys Gln Ser Ile Glu Asn Glu Leu Asn Ser Ala Asn Asp305 310
315 320Asn Pro Ile Ile Asp Ala Glu Gly Glu His Val Leu His Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Gly Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Pro
Ala Asn Leu Ser Gly Met Glu 370 375 380Gly Pro Arg Ala Ala Ile Asn
His Gly Leu Lys Ala Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Val Ala Ala Leu Leu Ile Thr Val Gln Gln Gly Val
Trp Leu 450 455 460Arg Arg Lys Leu Asn Ser Ser Val Thr Pro Glu Pro
Ser Val Glu Ala465 470 475 480Met Leu Gly Ala Leu Ser Ala Asp Val
Arg Met Ile Glu Glu Asp Arg 485 490 495Arg Leu Asp Pro Asp Leu Lys
Leu Leu Leu Glu Arg Ile Arg Val Arg 500 505 510Ser Trp Lys Leu Tyr
Asp 51571545DNAUnknownObtained from environmental sample
7atgacgacgc atcagcatga gccagtaacc ttcggccaaa ccccattgcg cattgaagac
60gtgctggctc tggccaatcg tcaggtgccg acccgtttgc aagacgacgc cgagtaccgc
120gaacgcatcg ccaaaggcgc gcgttttctc gattcattgc tcgacaaaga
gggcgtgatt 180tacggcgtga ccaccggcta cggcgactcg tgcgtggtgg
cggtgccgct ggagcatgtc 240gaagcgctgc cgcgtcacct ctacaccttc
cacggctgcg gcctgggcaa aatgcttgat 300gcccaggcca cccgcgccgt
gctggccgcc cgcttgcagt cgctgtgcca cggggtgtcc 360ggggtgcggg
ttgaactgct ggagcgcctg caagggttta tcgcccacga cattttgccg
420ctcatccccg aagagggctc ggtaggcgcc agcggtgacc tgacgccgct
gtcttatgtg 480gcggccacct tgtcgggcga gcgcgaggtc atgttcaacg
gcgaacgccg cctggcggct 540gacgtgcacc gcgaactgga ctggacgccg
ctggtgctgc ggcccaagga agcgctggcg 600ctgatgaacg gcacggccgt
aatgaccggc cttgcgtgcc tggcgtacgc ccgcgccgat 660tacctgctca
agctggcaac ccgcatcact gcactcaacg tggtggcgtt gcaaggtaac
720cctgaacact tcgatgagcg cctgttcgct gccaagccgc atccggggca
gatgcaagtg 780gcggcatggt tgcgccagga cctcgccatc gacgccccga
ccgcgccgct gcatcgcctg 840caagaccgtt actcgctgcg ctgcgcgccc
catgtgttgg gtgtgctggc tgacagcctg 900agctggctgc gcgggtttat
cgaagtcgaa ctcaacagcg ccaacgacaa cccgatcatc 960gacgccgaag
aagaacgcgt gctgcacggc gggcacttct atggcgggca cattgccttc
1020gccatggaca gcctcaaaac cctggtggcc aacgtggccg atctgcttga
tcgccaactc 1080gcgctgttgg tagacgtacg ttacaaccac ggattgccaa
gcaacctctc gggcgccagc 1140gccgaacggg cgatgctcaa ccacggcttc
aaagcggtgc agatcggcgc cagcgcctgg 1200accgccgaag ccctgaaaaa
cacgatgccg gccagcgtct tctcgcgctc aaccgaatgc 1260cacaaccagg
acaaagtgag catgggcacc atcgccgccc gtgacgccat tcgtgtgctg
1320gagctgaccg agcaagtggc cgccgcgacc ttgctggctg cccaccaggg
tgtgtggctg 1380cgcagccgcg acgcagacgc ccgcccgcta cccaccgcac
tggccgacat gcacgctgaa 1440ctggccaagg atttcgcccc tgtcattgaa
gaccgcgcac tggaagccga actgcgcctg 1500tgcctggccc gtattgccca
acaacactgg aggctgcatg cgtag 15458514PRTUnknownObtained from
environmental sample 8Met Thr Thr His Gln His Glu Pro Val Thr Phe
Gly Gln Thr Pro Leu1 5 10 15Arg Ile Glu Asp Val Leu Ala Leu Ala Asn
Arg Gln Val Pro Thr Arg 20 25 30Leu Gln Asp Asp Ala Glu Tyr Arg Glu
Arg Ile Ala Lys Gly Ala Arg 35 40 45Phe Leu Asp Ser Leu Leu Asp Lys
Glu Gly Val Ile Tyr Gly Val Thr 50 55 60Thr Gly Tyr Gly Asp Ser Cys
Val Val Ala Val Pro Leu Glu His Val65 70 75 80Glu Ala Leu Pro Arg
His Leu Tyr Thr Phe His Gly Cys Gly Leu Gly 85 90 95Lys Met Leu Asp
Ala Gln Ala Thr Arg Ala Val Leu Ala Ala Arg Leu 100 105 110Gln Ser
Leu Cys His Gly Val Ser Gly Val Arg Val Glu Leu Leu Glu 115 120
125Arg Leu Gln Gly Phe Ile Ala His Asp Ile Leu Pro Leu Ile Pro Glu
130 135 140Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser
Tyr Val145 150 155 160Ala Ala Thr Leu Ser Gly Glu Arg Glu Val Met
Phe Asn Gly Glu Arg 165 170 175Arg Leu Ala Ala Asp Val His Arg Glu
Leu Asp Trp Thr Pro Leu Val 180 185 190Leu Arg Pro Lys Glu Ala Leu
Ala Leu Met Asn Gly Thr Ala Val Met 195 200 205Thr Gly Leu Ala Cys
Leu Ala Tyr Ala Arg Ala Asp Tyr Leu Leu Lys 210 215 220Leu Ala Thr
Arg Ile Thr Ala Leu Asn Val Val Ala Leu Gln Gly Asn225 230 235
240Pro Glu His Phe Asp Glu Arg Leu Phe Ala Ala Lys Pro His Pro Gly
245 250 255Gln Met Gln Val Ala Ala Trp Leu Arg Gln Asp Leu Ala Ile
Asp Ala 260 265 270Pro Thr Ala Pro Leu His Arg Leu Gln Asp Arg Tyr
Ser Leu Arg Cys 275 280 285Ala Pro His Val Leu Gly Val Leu Ala Asp
Ser Leu Ser Trp Leu Arg 290 295 300Gly Phe Ile Glu Val Glu Leu Asn
Ser Ala Asn Asp Asn Pro Ile Ile305 310 315 320Asp Ala Glu Glu Glu
Arg Val Leu His Gly Gly His Phe Tyr Gly Gly 325 330 335His Ile Ala
Phe Ala Met Asp Ser Leu Lys Thr Leu Val Ala Asn Val 340 345 350Ala
Asp Leu Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Val Arg Tyr 355 360
365Asn His Gly Leu Pro Ser Asn Leu Ser Gly Ala Ser Ala Glu Arg Ala
370 375 380Met Leu Asn His Gly Phe Lys Ala Val Gln Ile Gly Ala Ser
Ala Trp385 390 395 400Thr Ala Glu Ala Leu Lys Asn Thr Met Pro Ala
Ser Val Phe Ser Arg 405 410 415Ser Thr Glu Cys His Asn Gln Asp Lys
Val Ser Met Gly Thr Ile Ala 420 425 430Ala Arg Asp Ala Ile Arg Val
Leu Glu Leu Thr Glu Gln Val Ala Ala 435 440 445Ala Thr Leu Leu Ala
Ala His Gln Gly Val Trp Leu Arg Ser Arg Asp 450 455 460Ala Asp Ala
Arg Pro Leu Pro Thr Ala Leu Ala Asp Met His Ala Glu465 470 475
480Leu Ala Lys Asp Phe Ala Pro Val Ile Glu Asp Arg Ala Leu Glu Ala
485 490 495Glu Leu Arg Leu Cys Leu Ala Arg Ile Ala Gln Gln His Trp
Arg Leu 500 505 510His Ala91545DNAUnknownObtained from
environmental sample 9atgacgacgc caacccatga gccggtaacc ttcggcgaac
gccctttgcg catcgaagac 60gtgctggccc tggccaaccg tcaggcaccc gtgcagttgc
agagcgacgc cgactaccgt 120gaacgcatcg ccaaaggtgc gcggttcctc
gattcgttgc tggacaagga aggtgtgatc 180tacggcgtca ccaccggcta
cggcgactct tgcgtggtcg cggtgccgtt gcatcacgtc 240gaggccctgc
cgcgtcatct ctacaccttc catggctgcg ggctgggcaa actgctcgac
300gctcaggcca cccgtgcggt gctggcggcg cgtttgcagt cgctgtgcca
cggcgtgtcc 360ggggtgcgca tcgagctgct ggaacgtctg catgccttcc
tcgaacacga catcctgccg 420ctgatccccg aagagggttc ggtgggcgcc
agcggcgatc tgacgccgct gtcctacgtc 480gccgcgactc tgtccggcga
gcgtgaagtg atgttccgtg gcgaacgccg ccagtccgcc 540gatgtgcatc
gcgaactcgg ttggacgccg ctggtgctgc gcccgaaaga agcgctggca
600ctgatgaacg gcaccgccgt gatgaccggc ctcgcctgcc tcgcttacgc
ccgcgccgat 660tacctgctgc aactggccac acgcatcacc gcgccgaacg
tggtggcgct gcaaggcaat 720ccggagcact tcgacgagcg cctgttcgcc
gccaagccgc atccgggtca aatgcaggtc 780gccgcgtggc tgcgcaagga
tttggcgatc gacgcaccga ccgcgccgtt gcatcgcctg 840caggatcgct
actcgctgcg ctgcgcaccg cacgtgctcg gcgtattggc cgacagcctg
900aactggctgc gttcgttcat cgagaccgaa ctcaacagcg ccaatgacaa
cccgatcatc 960gacgccgaag ccgagcgcgt gctgcacggc gggcacttct
acggcgggca tatcgcgttc 1020gccatggaca gcctgaaaaa cctcgtggcc
aacgtcgccg acctgcttga ccggcaactc 1080gcgctgctgg tggacgagcg
ttacaaccac ggtttgccga gcaacctgtc gggcgccagc 1140gccgaccgtg
cgatgctcaa ccacggcttc aaggccgtgc agatcggcgc cagcgcctgg
1200actgccgaag cgttgaaaaa caccatgccg gccagcgtgt tctcgcgctc
caccgagtgc 1260cacaaccagg acaaggtcag catgggcacc atcgccgccc
gcgacgccat tcgtgtgctg 1320gagctgaccg aacaggtggc cgccgccacg
ctgctggcgg ccaatcaggg cgtgtggctg 1380cgcagccgcg atgaagacgc
gcgtccactg ccaccgtccc tggcggcgat gcacgaacaa 1440ctggcccagg
acttcccgcc ggtgatcgaa gaccgcgccc tggaaggcga actgcacctg
1500tgcctgcaac gcatcgccga gcaacactgg aggctgcatg cgtag
154510514PRTUnknownObtained from environmental sample 10Met Thr Thr
Pro Thr His Glu Pro Val Thr Phe Gly Glu Arg Pro Leu1 5 10 15Arg Ile
Glu Asp Val Leu Ala Leu Ala Asn Arg Gln Ala Pro Val Gln 20 25 30Leu
Gln Ser Asp Ala Asp Tyr Arg Glu Arg Ile Ala Lys Gly Ala Arg 35 40
45Phe Leu Asp Ser Leu Leu Asp Lys Glu Gly Val Ile Tyr Gly Val Thr
50 55 60Thr Gly Tyr Gly Asp Ser Cys Val Val Ala Val Pro Leu His His
Val65 70 75 80Glu Ala Leu Pro Arg His Leu Tyr Thr Phe His Gly Cys
Gly Leu Gly 85 90 95Lys Leu Leu Asp Ala Gln Ala Thr Arg Ala Val Leu
Ala Ala Arg Leu 100 105 110Gln Ser Leu Cys His Gly Val Ser Gly Val
Arg Ile Glu Leu Leu Glu 115 120 125Arg Leu His Ala Phe Leu Glu His
Asp Ile Leu Pro Leu Ile Pro Glu 130 135 140Glu Gly Ser Val Gly Ala
Ser Gly Asp Leu Thr Pro Leu Ser Tyr Val145 150 155 160Ala Ala Thr
Leu Ser Gly Glu Arg Glu Val Met Phe Arg Gly Glu Arg 165 170 175Arg
Gln Ser Ala Asp Val His Arg Glu Leu Gly Trp Thr Pro Leu Val 180 185
190Leu Arg Pro Lys Glu Ala Leu Ala Leu Met Asn Gly Thr Ala Val Met
195 200 205Thr Gly Leu Ala Cys Leu Ala Tyr Ala Arg Ala Asp Tyr Leu
Leu Gln 210 215 220Leu Ala Thr Arg Ile Thr Ala Pro Asn Val Val Ala
Leu Gln Gly Asn225 230 235 240Pro Glu His Phe Asp Glu Arg Leu Phe
Ala Ala Lys Pro His Pro Gly 245 250 255Gln Met Gln Val Ala Ala Trp
Leu Arg Lys Asp Leu Ala Ile Asp Ala 260 265 270Pro Thr Ala Pro Leu
His Arg Leu Gln Asp Arg Tyr Ser Leu Arg Cys 275 280 285Ala Pro His
Val Leu Gly Val Leu Ala Asp Ser Leu Asn Trp Leu Arg 290 295 300Ser
Phe Ile Glu Thr Glu Leu Asn Ser Ala Asn Asp Asn Pro Ile Ile305 310
315 320Asp Ala Glu Ala Glu Arg Val Leu His Gly Gly His Phe Tyr Gly
Gly 325 330 335His Ile Ala Phe Ala Met Asp Ser Leu Lys Asn Leu Val
Ala Asn Val 340 345 350Ala Asp Leu Leu Asp Arg Gln Leu Ala Leu Leu
Val Asp Glu Arg Tyr 355 360 365Asn His Gly Leu Pro Ser Asn Leu Ser
Gly Ala Ser Ala Asp Arg Ala 370 375 380Met Leu Asn His Gly Phe Lys
Ala Val Gln Ile Gly Ala Ser Ala Trp385 390 395 400Thr Ala Glu Ala
Leu Lys Asn Thr Met Pro Ala Ser Val Phe Ser Arg 405 410 415Ser Thr
Glu Cys His Asn Gln Asp Lys Val Ser Met Gly Thr Ile Ala 420 425
430Ala Arg Asp Ala Ile Arg Val Leu Glu Leu Thr Glu Gln Val Ala Ala
435 440 445Ala Thr Leu Leu Ala Ala Asn Gln Gly Val Trp Leu Arg Ser
Arg Asp 450 455 460Glu Asp Ala Arg Pro Leu Pro Pro Ser Leu Ala Ala
Met His Glu Gln465 470 475 480Leu Ala Gln Asp Phe Pro Pro Val Ile
Glu Asp Arg Ala Leu Glu Gly 485 490 495Glu Leu His Leu Cys Leu Gln
Arg Ile Ala Glu Gln His Trp Arg Leu 500 505 510His Ala
111566DNAUnknownObtained from environmental sample 11atgcaacccg
ctgacgctgt gacttcttcc accccaaggc aagccgttcg cttcgaccag 60gggcgcctga
cgattgaaga catcgtcgcc attgcgcgca gaagcgccgg cgtcgaactc
120tcggccgacc cggcatttcg cgcggcgatt gccaagggcg ccgacttcct
cgaccgcctg 180ctgcgcgaag acggcaccat ctatggcgtg accacgggct
atggcgactc gtgcaccgtg 240accgtgccgc cggaactggt ggccgaattg
cctcatcacc tctataccta tcatggctgc 300ggcctcggtg aaaatttcac
gccggaacag acgcgcgcca tcatggctgc gcgcctggct 360tcgctgtcca
agggtttttc gggtgtgtcg gttgaattgc tggaacagat cgtcaagctg
420atgcagcacg acctgctgcc cgtgatgcct tcggaaggct cggtcggcgc
cagtggcgac 480ctgacgccct tgtcctacct ggccgcggtg ctctgcggcg
agcgtgaagt ctggcagggc 540ggcaagcagg tcgacgccgc ggacgccttg
cgcgcggccg gcattacgcc actgcgcctg 600cgtcccaagg aaggcctggc
catcatgaac ggcacggccg tcatgacggc gctggcctgc 660ctggcctacg
accgcgccga atacctgacc cgcctgtgca cgcgcatcac ggcgctggcc
720tcgttcgcgc tggacggcaa tgcgcatcac ttcaatgaaa ccctgttctc
ggtcaagccg 780caccccggca tgcagcaagt cgcggcctgg ctgcgccatg
acctgccgac cgatgtggtg 840gaacgcaacg gcaagcgcct gcaagaccgc
tattcgatcc gctgtgcgcc gcacgtgatc 900ggcgtgctgg ccgatgcctt
gcccttcctg cgccagtcca ttgaaaatga actcaacagc 960gccaacgaca
accccatcat cgatgcagaa ggcgagcacg tgctgcacgg cggccatttc
1020tacggcgggc atatcgcctt tgccatggac ggcatgaaaa atgccgtggc
caacctggcc 1080gacctgctcg accgccagat ggccctgctg gtcgatgcgc
gctacaacca cggcttgccg 1140gccaaccttt ccggcgccga aggcccgcgc
gctgcgatca accatggttt gaaagccttg 1200cagatcagcg cttcggcctg
gactgcggaa gcgctcaagc tgaccatgcc ggcttcggtg 1260ttttcgcgtt
ccacggaatg ccacaaccag gacaaggtca gcatgggcac catcgccgcg
1320cgcgattgcc tgcgcgtgct ggaattggtc gagcaggttg ttgcggcact
cttgatcacg 1380gtgcgccggg gcgtgtggct gcgccagaag gtacagcccg
atcaccagct ccacgccgct 1440ttgacggcca tgatggtgga actggaagcc
gatgtgccgg caatccgcga gggccgccgc 1500ctggaaccgg acttgcgcct
gatggtcgaa cgcatccgtt cccaagcctg gagcttgtat 1560ggctaa
156612521PRTUnknownObtained from environmental sample 12Met Gln Pro
Ala Asp Ala Val Thr Ser Ser Thr Pro Arg Gln Ala Val1 5 10 15Arg Phe
Asp Gln Gly Arg Leu Thr Ile Glu Asp Ile Val Ala Ile Ala 20 25 30Arg
Arg Ser Ala Gly Val Glu Leu Ser Ala Asp Pro Ala Phe Arg Ala 35 40
45Ala Ile Ala Lys Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Asp
50 55 60Gly Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val65 70 75 80Thr Val Pro Pro Glu Leu Val Ala Glu Leu Pro His His
Leu Tyr Thr 85 90 95Tyr His Gly Cys Gly Leu Gly Glu Asn Phe Thr Pro
Glu Gln Thr Arg 100 105 110Ala Ile Met Ala Ala Arg Leu Ala Ser Leu
Ser Lys Gly Phe Ser Gly 115 120 125Val Ser Val Glu Leu Leu Glu Gln
Ile Val Lys Leu Met Gln His Asp 130 135 140Leu Leu Pro Val Met Pro
Ser Glu Gly Ser Val Gly Ala Ser Gly Asp145 150 155 160Leu Thr Pro
Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Arg Glu 165 170 175Val
Trp Gln Gly Gly Lys Gln Val Asp Ala Ala Asp Ala Leu Arg Ala 180 185
190Ala Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile
195 200 205Met Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala
Tyr Asp 210 215 220Arg Ala Glu Tyr Leu Thr Arg Leu Cys Thr Arg Ile
Thr Ala Leu Ala225 230 235 240Ser Phe Ala Leu Asp Gly Asn Ala His
His Phe Asn Glu Thr Leu Phe 245 250 255Ser Val Lys Pro His Pro Gly
Met Gln Gln Val Ala Ala Trp Leu Arg 260 265 270His Asp Leu Pro Thr
Asp Val Val Glu Arg Asn Gly Lys Arg Leu Gln 275 280 285Asp Arg Tyr
Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala 290 295 300Asp
Ala Leu Pro Phe Leu Arg Gln Ser Ile Glu Asn Glu Leu Asn Ser305 310
315 320Ala Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu His Val Leu
His 325 330 335Gly Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met
Asp Gly Met 340 345 350Lys Asn Ala Val Ala Asn Leu Ala Asp Leu Leu
Asp Arg Gln Met Ala 355 360 365Leu Leu Val Asp Ala Arg Tyr Asn His
Gly Leu Pro Ala Asn Leu Ser 370 375 380Gly Ala Glu Gly Pro Arg Ala
Ala Ile Asn His Gly Leu Lys Ala Leu385 390 395 400Gln Ile Ser Ala
Ser Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met 405 410 415Pro Ala
Ser Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys 420 425
430Val Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu
435 440 445Leu Val Glu Gln Val Val Ala Ala Leu Leu Ile Thr Val Arg
Arg Gly 450 455 460Val Trp Leu Arg Gln Lys Val Gln Pro Asp His Gln
Leu His Ala Ala465 470 475 480Leu Thr Ala Met Met Val Glu Leu Glu
Ala Asp Val Pro Ala Ile Arg 485 490 495Glu Gly Arg Arg Leu Glu Pro
Asp Leu Arg Leu Met Val Glu Arg Ile 500 505 510Arg Ser Gln Ala Trp
Ser Leu Tyr Gly 515 520131566DNAUnknownObtained from environmental
sample 13atgcatcccg ttgacttgaa gaagacctca acctcagtcc gtacgattca
attcgacgat 60ggacggctga aaatcgagga tattgtcgat atcgcggaag gctcggctcg
ggtagcactg 120tccgatgctt ctgaattccg ttccgccatt gcgcgagggg
cggacttttt ggatcgtctg 180ctgcgcgaag agggcacgat ttatggtgtc
agcacgggct acggcgattc gtgcacggtg 240acggtcccgc ttgagctggt
cgccgagttg ccgcgccatc tctacactta tcacggctgc 300gggttggggg
agtatttaac tccggtacag acgcgggccg tgatcgccac ccgcctcaca
360tcgttgcgca aaggattctc tggcgtcagt ctggaactgc tgcatcaact
cgtgaggttg 420ctgcagtacg atttattgcc attgattcca tctgaaggtt
cggtgggcgc cagcggcgat 480ctcactccgc tatcctatct cgccgccgtg
ttgtgcggcg agggtgaagt ttggcgcaac 540ggcgtccatg taagcgccgc
gcacgcactg gctgaggccg gtatcgcacc gctgcggttg 600aggccaaaag
aaggactggc gatcatgaac gggacgtccg tcatgaccgc tttggcgtgc
660cttgcttatg cgcgggccga ttacctcacg cggctagtta ctcgcatcac
cgcattggcg 720tcgtttgcgc tggacggcaa tgcgcaccat ttcgatgcta
cgttgttctc agtgaagccg 780catccaggcc agcagcgggt ggccagttgg
ttgcgtcaag acttggcgtg cgatcagctg 840gaccccaacg gaaaacgact
ccaggatcgc tactcgattc gttgcgcgcc ccacgtgatc 900ggtgttctcg
ccgatgcgct gccatggttg cgcgagtccg tcgaaaatga actcaacagc
960gccaacgata atcccatcat cgacgcggag ggtgaacggg tgctctatgg
cggtcacttc 1020tatggcgggc atattggatt tgccatggac agcatgaaaa
atgccgtcgc aaacctggcg 1080gatctgcttg accggcagat ggcgctactc
gtcgacagcc gctacagcaa cggcttgccg 1140gccaacttat ctggggttca
agggccacgt gcagcgatca atcatggcct caaggggttg 1200cagatcagta
cttcggcatg gaccgcggag acgctcaagt
tgaccatgcc ggcgtcggtg 1260ttctcgcgtt cgaccgagtg ccacaaccag
gacaaagtca gcatgggcac cattgcggca 1320cgcgattgtt tgcgggtgct
cgaattgacg gagcaagtcg ccgcggcgct gctgattacc 1380gtgcgacagg
gagtctggct ccgttgcagg ctgcatcgat ccgtcgttcc cgaggcgacg
1440ttgaaggaca tgatggacgc cttgggcgct gatatcgctc agattgagga
ggaccgcagg 1500ctagagccgg atttgcgatt actgctggag cgcatccgcg
accgtgtctg gaaattgtat 1560gaataa 156614521PRTUnknownObtained from
environmental sample 14Met His Pro Val Asp Leu Lys Lys Thr Ser Thr
Ser Val Arg Thr Ile1 5 10 15Gln Phe Asp Asp Gly Arg Leu Lys Ile Glu
Asp Ile Val Asp Ile Ala 20 25 30Glu Gly Ser Ala Arg Val Ala Leu Ser
Asp Ala Ser Glu Phe Arg Ser 35 40 45Ala Ile Ala Arg Gly Ala Asp Phe
Leu Asp Arg Leu Leu Arg Glu Glu 50 55 60Gly Thr Ile Tyr Gly Val Ser
Thr Gly Tyr Gly Asp Ser Cys Thr Val65 70 75 80Thr Val Pro Leu Glu
Leu Val Ala Glu Leu Pro Arg His Leu Tyr Thr 85 90 95Tyr His Gly Cys
Gly Leu Gly Glu Tyr Leu Thr Pro Val Gln Thr Arg 100 105 110Ala Val
Ile Ala Thr Arg Leu Thr Ser Leu Arg Lys Gly Phe Ser Gly 115 120
125Val Ser Leu Glu Leu Leu His Gln Leu Val Arg Leu Leu Gln Tyr Asp
130 135 140Leu Leu Pro Leu Ile Pro Ser Glu Gly Ser Val Gly Ala Ser
Gly Asp145 150 155 160Leu Thr Pro Leu Ser Tyr Leu Ala Ala Val Leu
Cys Gly Glu Gly Glu 165 170 175Val Trp Arg Asn Gly Val His Val Ser
Ala Ala His Ala Leu Ala Glu 180 185 190Ala Gly Ile Ala Pro Leu Arg
Leu Arg Pro Lys Glu Gly Leu Ala Ile 195 200 205Met Asn Gly Thr Ser
Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Ala 210 215 220Arg Ala Asp
Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu Ala225 230 235
240Ser Phe Ala Leu Asp Gly Asn Ala His His Phe Asp Ala Thr Leu Phe
245 250 255Ser Val Lys Pro His Pro Gly Gln Gln Arg Val Ala Ser Trp
Leu Arg 260 265 270Gln Asp Leu Ala Cys Asp Gln Leu Asp Pro Asn Gly
Lys Arg Leu Gln 275 280 285Asp Arg Tyr Ser Ile Arg Cys Ala Pro His
Val Ile Gly Val Leu Ala 290 295 300Asp Ala Leu Pro Trp Leu Arg Glu
Ser Val Glu Asn Glu Leu Asn Ser305 310 315 320Ala Asn Asp Asn Pro
Ile Ile Asp Ala Glu Gly Glu Arg Val Leu Tyr 325 330 335Gly Gly His
Phe Tyr Gly Gly His Ile Gly Phe Ala Met Asp Ser Met 340 345 350Lys
Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln Met Ala 355 360
365Leu Leu Val Asp Ser Arg Tyr Ser Asn Gly Leu Pro Ala Asn Leu Ser
370 375 380Gly Val Gln Gly Pro Arg Ala Ala Ile Asn His Gly Leu Lys
Gly Leu385 390 395 400Gln Ile Ser Thr Ser Ala Trp Thr Ala Glu Thr
Leu Lys Leu Thr Met 405 410 415Pro Ala Ser Val Phe Ser Arg Ser Thr
Glu Cys His Asn Gln Asp Lys 420 425 430Val Ser Met Gly Thr Ile Ala
Ala Arg Asp Cys Leu Arg Val Leu Glu 435 440 445Leu Thr Glu Gln Val
Ala Ala Ala Leu Leu Ile Thr Val Arg Gln Gly 450 455 460Val Trp Leu
Arg Cys Arg Leu His Arg Ser Val Val Pro Glu Ala Thr465 470 475
480Leu Lys Asp Met Met Asp Ala Leu Gly Ala Asp Ile Ala Gln Ile Glu
485 490 495Glu Asp Arg Arg Leu Glu Pro Asp Leu Arg Leu Leu Leu Glu
Arg Ile 500 505 510Arg Asp Arg Val Trp Lys Leu Tyr Glu 515
520151563DNAUnknownObtained from environmental sample 15atgcccgttg
acttgaagaa gagctcaacc tcagtccgta cgattcaatt cgaccatggt 60cggctgaaaa
tcgaggatat tgtcgatatc tcggaagggt cggttcgcgt agcactgtct
120aatgcttctg aatttcgttc cgccattgcg cgaggggcgg actttttgga
tcgtctgctg 180cgcgaagagg gcacgatcta tggcgtcacc accggctacg
gcgattcatg caccgtggcg 240gtcccgcttg agctggtcgc cgagttgccg
cgccagctct atgtctatca cggctgtgga 300ctgggcgagt ctttgagtcc
gctacagacg cgggccgtga tcgccacccg cctcacatcg 360ttgtgcaaag
gactctctgg cgtcagtctg gaactgctgc atcaactcgt gaggttgctg
420cagtacgatt tattgccatt gattccatct gaaggttcgg tgggggctag
cggcgatctc 480actccgctat cctatctcgc tgccgtgttg tgcggcgagg
gcgaagtttg gcgcaacggc 540gtccatgtaa gcgcagcgga cgcactggct
gaggccggta tcgcaccgct gcggttgagg 600ccaaaagaag gactggcgat
catgaatggg acgtcggtca tgaccgcttt ggcgtgcctt 660gcttatgggc
gcgccgatta cctcacgcgg ctagttactc gcatcacggc attggcgtcg
720tttgcgctgg acggcaatgc gcatcatttc gatgccacgt tgttctcagt
gaagccgcat 780ccaggccagc agcgggtggc cggttggttg cgtcaagact
tggcgtgcga tcagctggat 840cccaacggaa agcgcctcca agatcgctac
tcgatccgtt gcgccccaca cgtgatcggt 900gttctcgccg atgccctgcc
atggttgcgc gactccatcg aaaacgaact caacagcgcc 960aacgacaatc
cgatcatcga tgcggagggc gaacgcgtgc tctatggcgg tcacttctat
1020ggggggcata ttggatttgc catggacagc atgaaaaatg ccgtcgccaa
cctggcggat 1080ctgcttgacc ggcagatggc actgctggtc gacagccgct
atagcaacgg cttgccggcc 1140aatttatctg gggttcaagg gccacgtgcg
gcgatcaatc atgggctcaa ggggctgcag 1200atcagtgttt cggcatggac
ggcagaagcc ctcaagttga ccatgccggc gtcagtgttc 1260tcgcgttcga
ccgagtgcca caaccaggac aaagtcagca tgggtaccat tgcggcacgc
1320gattgtttgc gtgtgctcga attgactgag caagtcgccg cggcgctgct
gatcaccgtg 1380cgacagggag tctggctccg ttgcaggctg aatcgatccg
tcgctccgga ggcgacgttg 1440aagaacatga ttgacgccct gggcgctgat
atcgctgaga ttgaggagga ccgcaggcta 1500gagccagatt tgcgattact
gctggagcgg atccgccacc gtgcctggaa attgtatgaa 1560taa
156316520PRTUnknownObtained from environmental sample 16Met Pro Val
Asp Leu Lys Lys Ser Ser Thr Ser Val Arg Thr Ile Gln1 5 10 15Phe Asp
His Gly Arg Leu Lys Ile Glu Asp Ile Val Asp Ile Ser Glu 20 25 30Gly
Ser Val Arg Val Ala Leu Ser Asn Ala Ser Glu Phe Arg Ser Ala 35 40
45Ile Ala Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Ala65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Ser Leu Ser Pro Leu
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Leu Ser Gly Val 115 120 125Ser Leu Glu Leu Leu His Gln Leu
Val Arg Leu Leu Gln Tyr Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val His Val Ser Ala Ala Asp Ala Leu Ala Glu Ala 180 185
190Gly Ile Ala Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Gly Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Ala Cys Asp
Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Asp Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Arg Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Gly Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Val Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Thr Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Val Ala Pro
Glu Ala Thr Leu465 470 475 480Lys Asn Met Ile Asp Ala Leu Gly Ala
Asp Ile Ala Glu Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510His Arg Ala Trp Lys
Leu Tyr Glu 515 520171545DNAUnknownObtained from environmental
sample 17atgacgacgc caacgcttga gccggtaacc ttcggcgaac tccctttgcg
catcgaagac 60gtgttggctt tggccaaccg tcaggtgccg acgcagttgc aaggcgatcc
cgagttccgt 120cggcgcatcg ccaagggcgc gcaattcctt gactcccttt
tggacaagga aggcgtgatc 180tatggcgtga ccaccggtta cggcgactcc
tgcgtggtag cggtgccgct gcatcacgtc 240gaagcgctgc cgcgtcatct
gtacaccttc catggctgcg ggctgggcaa gctgctcgat 300gcccaggcca
ctcgcgccgt gctggcggcg cgtttgcagt cgctgtgcca cggcgtatcc
360ggtgttcggg tggaattgct cgaacgcctg caagcgtttc tcgaacacga
catcctgccg 420ctgattccgg aagaaggctc ggtgggcgcc agcggcgatc
tgacgccgtt gtcctacatc 480gccgcgaccc tgtccggcga gcgcgaagtg
atgttccgcg gcgaacgtcg tcaagccgcc 540gatgtgcatc gcgaactcgg
ttggcagccg ctggtgctgc ggcccaagga agccctggcc 600ctgatgaacg
gcaccgccgt catgaccggc ctcgcttgcc tggcctacgc ccgtgccgat
660tacctgctgc aactggccac gcgcatcacc gcgctgaacg tggtcgccct
gcaaggcaat 720cccgagcatt tcgacgaacg cctgttcgcc gccaagccac
acccggggca gatgcaagtc 780gccgcgtggt tgcgcaagga cctggcgatc
gacgcgccaa ccgcgccgct gcatcgcctg 840caggaccgct attccctgcg
ctgcgcgccc cacgtcctcg gcgtgctggc cgacagcctg 900aactggctgc
gttcgttcat cgagatcgaa ctcaacagcg ccaacgacaa cccgatcatc
960gatgccgagg ccgagcgcgt gctgcacggc gggcactttt acggcggcca
catcgcgttc 1020gccatggaca gcctgaaaaa cctcgtggcc aacgtcgccg
acctgctgga tcgccaactc 1080gcgctgctgg tggacgagcg ttacaaccac
ggcctgccga gcaacctgtc cggcgccagc 1140gccgaccgcg cgatgctcaa
ccacggcttc aaggcggtgc agatcggcac cagcgcctgg 1200accgccgaag
cgttgaaaaa caccatgccg gccagcgtgt tctcgcgctc caccgagtgc
1260cacaaccagg acaaggtgag catgggcacc attgccgccc gcgatgcgat
ccgcgtactg 1320gaactgaccg aacaggtggc cgccgccacg ctgctggccg
ccaaccaggg cgtctggctg 1380cgtgcccagg ccgaagacgc ccggccgttg
ccaccggcgc tggcggccat gcacgaagca 1440ctggccaagg acttcccgcc
ggtcatcgaa gaccgcgcgc tggaaggcga actgcgcctg 1500tgcctgcaac
gtatcgctga acaacactgg aggctgcatg cgtag 154518514PRTUnknownObtained
from environmental sample 18Met Thr Thr Pro Thr Leu Glu Pro Val Thr
Phe Gly Glu Leu Pro Leu1 5 10 15Arg Ile Glu Asp Val Leu Ala Leu Ala
Asn Arg Gln Val Pro Thr Gln 20 25 30Leu Gln Gly Asp Pro Glu Phe Arg
Arg Arg Ile Ala Lys Gly Ala Gln 35 40 45Phe Leu Asp Ser Leu Leu Asp
Lys Glu Gly Val Ile Tyr Gly Val Thr 50 55 60Thr Gly Tyr Gly Asp Ser
Cys Val Val Ala Val Pro Leu His His Val65 70 75 80Glu Ala Leu Pro
Arg His Leu Tyr Thr Phe His Gly Cys Gly Leu Gly 85 90 95Lys Leu Leu
Asp Ala Gln Ala Thr Arg Ala Val Leu Ala Ala Arg Leu 100 105 110Gln
Ser Leu Cys His Gly Val Ser Gly Val Arg Val Glu Leu Leu Glu 115 120
125Arg Leu Gln Ala Phe Leu Glu His Asp Ile Leu Pro Leu Ile Pro Glu
130 135 140Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser
Tyr Ile145 150 155 160Ala Ala Thr Leu Ser Gly Glu Arg Glu Val Met
Phe Arg Gly Glu Arg 165 170 175Arg Gln Ala Ala Asp Val His Arg Glu
Leu Gly Trp Gln Pro Leu Val 180 185 190Leu Arg Pro Lys Glu Ala Leu
Ala Leu Met Asn Gly Thr Ala Val Met 195 200 205Thr Gly Leu Ala Cys
Leu Ala Tyr Ala Arg Ala Asp Tyr Leu Leu Gln 210 215 220Leu Ala Thr
Arg Ile Thr Ala Leu Asn Val Val Ala Leu Gln Gly Asn225 230 235
240Pro Glu His Phe Asp Glu Arg Leu Phe Ala Ala Lys Pro His Pro Gly
245 250 255Gln Met Gln Val Ala Ala Trp Leu Arg Lys Asp Leu Ala Ile
Asp Ala 260 265 270Pro Thr Ala Pro Leu His Arg Leu Gln Asp Arg Tyr
Ser Leu Arg Cys 275 280 285Ala Pro His Val Leu Gly Val Leu Ala Asp
Ser Leu Asn Trp Leu Arg 290 295 300Ser Phe Ile Glu Ile Glu Leu Asn
Ser Ala Asn Asp Asn Pro Ile Ile305 310 315 320Asp Ala Glu Ala Glu
Arg Val Leu His Gly Gly His Phe Tyr Gly Gly 325 330 335His Ile Ala
Phe Ala Met Asp Ser Leu Lys Asn Leu Val Ala Asn Val 340 345 350Ala
Asp Leu Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Glu Arg Tyr 355 360
365Asn His Gly Leu Pro Ser Asn Leu Ser Gly Ala Ser Ala Asp Arg Ala
370 375 380Met Leu Asn His Gly Phe Lys Ala Val Gln Ile Gly Thr Ser
Ala Trp385 390 395 400Thr Ala Glu Ala Leu Lys Asn Thr Met Pro Ala
Ser Val Phe Ser Arg 405 410 415Ser Thr Glu Cys His Asn Gln Asp Lys
Val Ser Met Gly Thr Ile Ala 420 425 430Ala Arg Asp Ala Ile Arg Val
Leu Glu Leu Thr Glu Gln Val Ala Ala 435 440 445Ala Thr Leu Leu Ala
Ala Asn Gln Gly Val Trp Leu Arg Ala Gln Ala 450 455 460Glu Asp Ala
Arg Pro Leu Pro Pro Ala Leu Ala Ala Met His Glu Ala465 470 475
480Leu Ala Lys Asp Phe Pro Pro Val Ile Glu Asp Arg Ala Leu Glu Gly
485 490 495Glu Leu Arg Leu Cys Leu Gln Arg Ile Ala Glu Gln His Trp
Arg Leu 500 505 510His Ala191557DNAUnknownObtained from
environmental sample 19atggatccca ttaaaacaaa aaaaccgtca cgcatcgttt
gcttcgccgc ggaccggctg 60acgatcgaag atatcgtcga tctcgctgat ggcacagcgc
acgccgcgtt gtccgaggag 120ccggcgtttc gcgcttttat cggccgcggc
gcggcctttc tagagcagcg gctgcatgat 180gacggttcgg tttacggtgt
aacgaccggt ttcggtgatt cctgcacagt ggccgtggcg 240cccgaactgg
tggacgagct gccgcgtcat ctttacacgt tccacggctg cggcttggga
300aagcatttta ctcccgtgga aacccgggcg atcgtcgcaa cccgtctggc
gtcgctgtgc 360aagggcttct cgggcgtcac tatagagctg ctcgagcagc
tgactcggct gttgcgccga 420gatatcctgc ccgccattcc cagtgaaggc
tccgtcgggg cgagcggcga tcttactccg 480ctgtcgtatc tcgccgccgt
ggtctgcggc gaaggcgagg tgtgggaagg cggtaagatt 540gttgccgcag
cgcctgtcct ggccggagcc ggtataaccg ctctccggtt gcgccctaaa
600gaagcgctcg cgatcatgaa cggcaccgcg gtgatgaccg cgctggcgtg
tctcgcctac 660gcgcgcgcgg agtatctggc ccggctggcg gcgcgaatta
cagcgctggc ctcgtttgct 720ctggatggga acgccgagca ttttgacgcc
gggttgttcg ccctcaaacc tcatcccggg 780caacagcgcg tggccgcctg
gctgcgcgcg gacttgccag atgggggcag cgcgctcaac 840ggcaagcggc
tacaggatcg ctactcgata aggtgctcgc cgcacgttat cggcgtgctg
900gtcgacgcct tgccgtggct gcggcagtcg atagagaacg aactcaacag
cgtcaacgat 960aatccggtgg tcgacggcgc cggcgaacgg gtcttgcatg
gcggccactt ctacggcggc 1020cacatcgcgt ttgccatgga cagcatgaaa
aacgcggtgg cgaatctggc ggatctgctc 1080gatcgacaga tggcgctgtt
ggtcgatgcc cgctacagca acggacttcc ggcgaatctc 1140tccggcgtcg
aggggccgcg cgcggcgatc aatcacggcc tcaaggcgct gcaaatcagc
1200gtttccgcgt ggtgcgccga ggcgctcaag ctgaccatgc cggcctcggt
gttttcgcgc 1260tccaccgagt gtcacaacca ggataaagtg agcatgggca
ccattgccgc gcgggattgt 1320ctccgggtct tggaattgac cgagcaagtc
gcggttgcgc tcttgatcac gacgcgccag 1380gccgtctggt ggcggcgccg
gttgagtcct tccctgaccc tctccgcgcc gctgcgggag 1440atgatcgacg
cgctcggcgc cgatatcgcg gtgctcgaag aagaccgccg gctggaaccg
1500gatctcaggc ggctcctgga gcgtttaaac gaccgtgcct ggaggctcta tgagtaa
155720518PRTUnknownObtained from environmental sample 20Met Asp Pro
Ile Lys Thr Lys Lys Pro Ser Arg Ile Val Cys Phe Ala1 5 10 15Ala Asp
Arg Leu Thr Ile Glu Asp Ile Val
Asp Leu Ala Asp Gly Thr 20 25 30Ala His Ala Ala Leu Ser Glu Glu Pro
Ala Phe Arg Ala Phe Ile Gly 35 40 45Arg Gly Ala Ala Phe Leu Glu Gln
Arg Leu His Asp Asp Gly Ser Val 50 55 60Tyr Gly Val Thr Thr Gly Phe
Gly Asp Ser Cys Thr Val Ala Val Ala65 70 75 80Pro Glu Leu Val Asp
Glu Leu Pro Arg His Leu Tyr Thr Phe His Gly 85 90 95Cys Gly Leu Gly
Lys His Phe Thr Pro Val Glu Thr Arg Ala Ile Val 100 105 110Ala Thr
Arg Leu Ala Ser Leu Cys Lys Gly Phe Ser Gly Val Thr Ile 115 120
125Glu Leu Leu Glu Gln Leu Thr Arg Leu Leu Arg Arg Asp Ile Leu Pro
130 135 140Ala Ile Pro Ser Glu Gly Ser Val Gly Ala Ser Gly Asp Leu
Thr Pro145 150 155 160Leu Ser Tyr Leu Ala Ala Val Val Cys Gly Glu
Gly Glu Val Trp Glu 165 170 175Gly Gly Lys Ile Val Ala Ala Ala Pro
Val Leu Ala Gly Ala Gly Ile 180 185 190Thr Ala Leu Arg Leu Arg Pro
Lys Glu Ala Leu Ala Ile Met Asn Gly 195 200 205Thr Ala Val Met Thr
Ala Leu Ala Cys Leu Ala Tyr Ala Arg Ala Glu 210 215 220Tyr Leu Ala
Arg Leu Ala Ala Arg Ile Thr Ala Leu Ala Ser Phe Ala225 230 235
240Leu Asp Gly Asn Ala Glu His Phe Asp Ala Gly Leu Phe Ala Leu Lys
245 250 255Pro His Pro Gly Gln Gln Arg Val Ala Ala Trp Leu Arg Ala
Asp Leu 260 265 270Pro Asp Gly Gly Ser Ala Leu Asn Gly Lys Arg Leu
Gln Asp Arg Tyr 275 280 285Ser Ile Arg Cys Ser Pro His Val Ile Gly
Val Leu Val Asp Ala Leu 290 295 300Pro Trp Leu Arg Gln Ser Ile Glu
Asn Glu Leu Asn Ser Val Asn Asp305 310 315 320Asn Pro Val Val Asp
Gly Ala Gly Glu Arg Val Leu His Gly Gly His 325 330 335Phe Tyr Gly
Gly His Ile Ala Phe Ala Met Asp Ser Met Lys Asn Ala 340 345 350Val
Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln Met Ala Leu Leu Val 355 360
365Asp Ala Arg Tyr Ser Asn Gly Leu Pro Ala Asn Leu Ser Gly Val Glu
370 375 380Gly Pro Arg Ala Ala Ile Asn His Gly Leu Lys Ala Leu Gln
Ile Ser385 390 395 400Val Ser Ala Trp Cys Ala Glu Ala Leu Lys Leu
Thr Met Pro Ala Ser 405 410 415Val Phe Ser Arg Ser Thr Glu Cys His
Asn Gln Asp Lys Val Ser Met 420 425 430Gly Thr Ile Ala Ala Arg Asp
Cys Leu Arg Val Leu Glu Leu Thr Glu 435 440 445Gln Val Ala Val Ala
Leu Leu Ile Thr Thr Arg Gln Ala Val Trp Trp 450 455 460Arg Arg Arg
Leu Ser Pro Ser Leu Thr Leu Ser Ala Pro Leu Arg Glu465 470 475
480Met Ile Asp Ala Leu Gly Ala Asp Ile Ala Val Leu Glu Glu Asp Arg
485 490 495Arg Leu Glu Pro Asp Leu Arg Arg Leu Leu Glu Arg Leu Asn
Asp Arg 500 505 510Ala Trp Arg Leu Tyr Glu
515211560DNAUnknownObtained from environmental sample 21atggaattca
tgcgtcaagg taatccgccc tcgcgcgtta tttgttttgg ctcggaccgg 60ttggcaattg
ccgatgtggt cgcgatcgcc gaaggcacgg cggcggcgat cctgtcggat
120gctccgcaat tccgcgcctt gattgcccgc ggcgccgatt ttctcgaccg
tctgctgcgc 180gacgaaggtg tgatctatgg cgtgacgacg ggttacggtg
attcctgcac ggtagctgtg 240ccgcccgagt tggtcgcgga actgccgcgc
catctctata cctttcacgg ctgcggtctg 300ggagcgcact tgacccccga
acagacccgc gctgtgatcg cgactcggct cgcctctttg 360tgcaaaggtt
tctccggggt gagcgttgaa ttgctggagc aactggtgcg gctgctgcgc
420cacgatatct tgccacgaat accttcggaa ggttcggtgg gcgccagcgg
cgacctcacg 480cccttgtcct accttgccgc ggtaatttgc ggcgagggcg
aggtttggcg cgacggcgcg 540tccgcgccgg ccgcgcaggc cttgctcgaa
gcggacatca cgccgctgca gttgcggccg 600aaggagggac tggcggtcat
gaacggcacc gcggtcatga ccgcgatggc ttgtttggcc 660tatgtccgcg
ccgagtacct tgtccgcctg gcgacgcgcg tctcggcgct cgcctccttt
720gctttggacg gcaatgccga gcattttgat gcgacgctat tcagcgtcaa
gccgcacgac 780gggctgcagc gggttgccgc atggttgcgc gacgacctgc
cgagccgggc ggcgccctac 840gatggaaaac gcctgcagga ccgctactcg
attcgctgcg ccccacacgt gatcggtgtg 900ctcgccgatg cccttggctg
gatgcgcgaa accatcgaaa acgagattaa tagcgccaac 960gacaacccga
ttatcgatgc cgataacgag cgggtcctgc acggcggcca tttctacggc
1020ggacatatcg cgttcgtcat ggatagcatg aaaaatgccg tcgccaattt
ggccgacatg 1080ctcgaccgcc aggtcgctct gctggtggat agccgtttta
acaatggcct gccggcgaac 1140ctgtccggtg tcgaagggcc gcgcgccgcg
atcaatcatg gcctcaaggc gctgcaaatc 1200agcgcgtcgg cttgggcggc
ggaagcgttg aaattgacca tgccggcgtc ggtgttctcc 1260cgttcgaccg
agtgccacaa ccaggacaag gtaagcatgg gcacgattgc cgcgcgcgat
1320tgcttgcgcg tcatcgagtt gaccgaacaa gtgaccgcgg cgtcattgat
cgccgcgcgc 1380caaggcgtat ggctgcgccg ccgatcaaat ccggccgcgg
tgccttcgac cgggctgcag 1440cgcatgatcg atgcgctgag cgacgatgtg
gccgtgatca tcgaagaccg ccggctcgag 1500gcggatttgc gctccctgct
cgagcgcatt cgtcagcgcg cttggaaacc gtatgagtga
156022519PRTUnknownObtained from environmental sample 22Met Glu Phe
Met Arg Gln Gly Asn Pro Pro Ser Arg Val Ile Cys Phe1 5 10 15Gly Ser
Asp Arg Leu Ala Ile Ala Asp Val Val Ala Ile Ala Glu Gly 20 25 30Thr
Ala Ala Ala Ile Leu Ser Asp Ala Pro Gln Phe Arg Ala Leu Ile 35 40
45Ala Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Asp Glu Gly Val
50 55 60Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val Ala
Val65 70 75 80Pro Pro Glu Leu Val Ala Glu Leu Pro Arg His Leu Tyr
Thr Phe His 85 90 95Gly Cys Gly Leu Gly Ala His Leu Thr Pro Glu Gln
Thr Arg Ala Val 100 105 110Ile Ala Thr Arg Leu Ala Ser Leu Cys Lys
Gly Phe Ser Gly Val Ser 115 120 125Val Glu Leu Leu Glu Gln Leu Val
Arg Leu Leu Arg His Asp Ile Leu 130 135 140Pro Arg Ile Pro Ser Glu
Gly Ser Val Gly Ala Ser Gly Asp Leu Thr145 150 155 160Pro Leu Ser
Tyr Leu Ala Ala Val Ile Cys Gly Glu Gly Glu Val Trp 165 170 175Arg
Asp Gly Ala Ser Ala Pro Ala Ala Gln Ala Leu Leu Glu Ala Asp 180 185
190Ile Thr Pro Leu Gln Leu Arg Pro Lys Glu Gly Leu Ala Val Met Asn
195 200 205Gly Thr Ala Val Met Thr Ala Met Ala Cys Leu Ala Tyr Val
Arg Ala 210 215 220Glu Tyr Leu Val Arg Leu Ala Thr Arg Val Ser Ala
Leu Ala Ser Phe225 230 235 240Ala Leu Asp Gly Asn Ala Glu His Phe
Asp Ala Thr Leu Phe Ser Val 245 250 255Lys Pro His Asp Gly Leu Gln
Arg Val Ala Ala Trp Leu Arg Asp Asp 260 265 270Leu Pro Ser Arg Ala
Ala Pro Tyr Asp Gly Lys Arg Leu Gln Asp Arg 275 280 285Tyr Ser Ile
Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala 290 295 300Leu
Gly Trp Met Arg Glu Thr Ile Glu Asn Glu Ile Asn Ser Ala Asn305 310
315 320Asp Asn Pro Ile Ile Asp Ala Asp Asn Glu Arg Val Leu His Gly
Gly 325 330 335His Phe Tyr Gly Gly His Ile Ala Phe Val Met Asp Ser
Met Lys Asn 340 345 350Ala Val Ala Asn Leu Ala Asp Met Leu Asp Arg
Gln Val Ala Leu Leu 355 360 365Val Asp Ser Arg Phe Asn Asn Gly Leu
Pro Ala Asn Leu Ser Gly Val 370 375 380Glu Gly Pro Arg Ala Ala Ile
Asn His Gly Leu Lys Ala Leu Gln Ile385 390 395 400Ser Ala Ser Ala
Trp Ala Ala Glu Ala Leu Lys Leu Thr Met Pro Ala 405 410 415Ser Val
Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser 420 425
430Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Ile Glu Leu Thr
435 440 445Glu Gln Val Thr Ala Ala Ser Leu Ile Ala Ala Arg Gln Gly
Val Trp 450 455 460Leu Arg Arg Arg Ser Asn Pro Ala Ala Val Pro Ser
Thr Gly Leu Gln465 470 475 480Arg Met Ile Asp Ala Leu Ser Asp Asp
Val Ala Val Ile Ile Glu Asp 485 490 495Arg Arg Leu Glu Ala Asp Leu
Arg Ser Leu Leu Glu Arg Ile Arg Gln 500 505 510Arg Ala Trp Lys Pro
Tyr Glu 515231563DNAUnknownObtained from environmental sample
23atgcatgccg ctgagtcaaa gacatcagcc ttacctcgga caattgtgtt cgaccatgga
60tggctgaaaa tcgaagacat cgtcgacatt gcggaaggtt cggctagggt cgcgttgtcc
120gaggctgctg catttcatgt tggcatcaag cgaggggcag acttcctgga
gcgtctgctc 180cgccaagagg gcacgatcta tggcgttact acgggttacg
gcgattcgtg cacggtgact 240gtccctcctg agatggtcgc cgagttgccg
cgccagctct acgtttatca cggctgcggg 300ctgggcgagt atctgagtcc
ggtgcagacg cgcgccgtga tggcgacccg cctcacgtcg 360ttgtgcaaag
ggttttccgg cgtgagcctg gaactcctgc aacaaatcgt gaagctactc
420cagcgcgatt tattgccgtt gattccgtcg gagggctcgg taggtgccag
tggcgacctg 480actccattat cgtatttcgc ggcggttttg tgcggcgagg
gcgacgtttg gcgcagcggc 540gtccatctca gggccactcg ggcactggcc
gaggccggca tcacaccgct gtgtctgaga 600cccaaagagg gcttggcgat
catgaatggg accgcggtca tgaccgcttt ggcctgcctt 660gcttatgtgc
gcgccgatta tctcacccga ctggtcactc ggatcaccgc gttggcgtcg
720tttgcgcttg acggcaatgc gcaccatttc gatgccgcgt tgttcgccgt
gaaaccgcat 780ccaggcatgc agcgagtggc cggttggttg cgtcaagact
tgttctcgga tcagttggac 840cccaacgaca agcgcctaca ggatcgctat
tcgatccgtt gcgcgcccca cgtgatcggt 900gttctagccg atgcgctgcc
gtggttgcgc gagtccattg aaaatgagct caacagcgcc 960aacgacaatc
ccatcatcga tgcggtaggc caaggcgtgt tgtgtggcgg tcacttttac
1020ggcgggcata tcgcatttgc tatggacagc atgaaaaatg ccgtcgccaa
cctggcggat 1080ctgctcgacc ggcagatggc gctgttggtg gatagccgct
acagcaatgg gctgccagcc 1140aacttgtctg gggttcaagg gccacgcgcc
ccgatcaacc atgggcttaa ggggttgcag 1200atcggcgcct cggcctggac
tgccgaagcg ctcaagttga ccatgccggc atcggtcttc 1260tcgcgttcga
ccgagtgcca taatcaggac aaagtcagcc tcggtactat tgccgcacgc
1320gattgtttgc gtgtgctcga attgacggag caagttgccg cggcgctgct
cattgccgtg 1380cggcaaggag tctggctgcg ttgccgactc aatcggtcct
tggctccggc gaagacgcta 1440aaaaacatga tggacgcgct agccgccgat
atccgcgtga tcgaggagga tcgcaagctg 1500gaaccggatt tgcgattact
gttggagcgc atccgcgacc gcttctggaa gctctatgaa 1560taa
156324520PRTUnknownObtained from environmental sample 24Met His Ala
Ala Glu Ser Lys Thr Ser Ala Leu Pro Arg Thr Ile Val1 5 10 15Phe Asp
His Gly Trp Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Glu Ala Ala Ala Phe His Val Gly 35 40
45Ile Lys Arg Gly Ala Asp Phe Leu Glu Arg Leu Leu Arg Gln Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Pro Glu Met Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Lys Leu Leu Gln Arg Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Phe Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Ser Gly Val His Leu Arg Ala Thr Arg Ala Leu Ala Glu Ala 180 185
190Gly Ile Thr Pro Leu Cys Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Ala Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Phe Ser Asp
Gln Leu Asp Pro Asn Asp Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Val Gly Gln Gly Val Leu Cys
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Pro
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Gly Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Leu Ala Pro
Ala Lys Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ala Ala
Asp Ile Arg Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Asp Arg Phe Trp Lys
Leu Tyr Glu 515 520251557DNAUnknownObtained from environmental
sample 25atggatccca ttaaaacaaa aaaaccgtca cgcatcgttt gcttcgccgc
ggaccggctg 60accatcgaag atatcgtcga tctcgctgat ggcacagcgc acgccgcgtt
gtccgaggag 120ccggcgtttc gcgcgtttat cggccgcggc gcggcctttc
tagagcagcg actgcgtgat 180gacggttcgg tttacggtgt aacgaccggt
ttcggtgatt cctgcacggt ggccgtggcg 240cccgaactgg tggacgagct
gccgcgtcat ctttacacgt tccacggctg cggcttggga 300aagcatttta
ctcccgtgga aacccgggcg atcgtcgcaa cccgtctggc gtcgctgtgc
360aagggcttct cgggcgtcac tatagagctg ctcgagcagc tgactcggct
gttgcgccga 420gatatcctgc ccgctattcc cagtgaaggc tccgtcgggg
cgagcggcga tcttactccg 480ctgtcgtatc tcgccgccgt ggtctgcggc
gagggcgagg tgtggcaagg cggtaagatt 540gttgccgcag cgcctgtcct
ggccggagcc ggcataaccg ctctccggtt gcgccctaaa 600gaagcgctcg
cgatcatgaa cggcaccgcg gtgatgaccg cgctggcgtg tctcgcctac
660tcgcgcgcgg agtatctggc ccggctggcg gcgcgaatta cggcgctggc
ctcgtttgct 720ctggatggga acgccgagca ttttgacgcc gggttgttcg
ccctcaaacc tcatcccggg 780caacaacgcg tggccgcctg gctgcgcgcg
gacttgcccg atgggggcag cgcgctcaac 840ggcaagcggc tacaggatcg
ctactcgata aggtgcgcgc cgcacgttat cggcgtgctg 900gtggacgcct
tgccgtggct gcggcagtcg atcgagaacg aactcaacag cgtcaacgat
960aatccggtgg tcgacggcgc cggcgaacgg gtcttgcatg gcggccactt
ctacggcggc 1020cacatcgcgt ttgccatgga cagcatgaaa aacgcggtgg
cgaatctggc ggatctgctc 1080gatcgacaga tggcactgtt ggtcgatgcc
cgctacagca acggactccc ggcgaatctc 1140tccggcgtcg aggggccgcg
cgcggcgatc aatcacggcc tcaaggcgct gcaaatcagc 1200gtttccgcgt
ggtgcgccga ggcgctcaag ctgaccatgc cggcctcggt gttttcgcgc
1260tccaccgagt gtcacaacca ggataaagtg agcatgggca ccattgccgc
gcgggattgt 1320ctccgggtct tggaattgac cgagcaagtc gcggttgcgc
tcttgatcac gacgcgccag 1380gccgtctggt ggcggcgccg gttgagtcct
tccctgaccc tctccgcgcc gctgcgggag 1440atgatcgacg cgctcggcgc
cgatatcgcg gtgctcgaag aagaccgccg gctggaaccg 1500gatctcaggc
ggctcctgga gcgtttaaac gaccgtgcct ggaggctcta tgagtaa
155726518PRTUnknownObtained from environmental sample 26Met Asp Pro
Ile Lys Thr Lys Lys Pro Ser Arg Ile Val Cys Phe Ala1 5 10 15Ala Asp
Arg Leu Thr Ile Glu Asp Ile Val Asp Leu Ala Asp Gly Thr 20 25 30Ala
His Ala Ala Leu Ser Glu Glu Pro Ala Phe Arg Ala Phe Ile Gly 35 40
45Arg Gly Ala Ala Phe Leu Glu Gln Arg Leu Arg Asp Asp Gly Ser Val
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Ala Val
Ala65 70 75 80Pro Glu Leu Val Asp Glu Leu Pro Arg His Leu Tyr Thr
Phe His Gly 85 90 95Cys Gly Leu Gly Lys His Phe Thr Pro Val Glu Thr
Arg Ala Ile Val 100 105 110Ala Thr Arg Leu Ala Ser Leu Cys Lys
Gly
Phe Ser Gly Val Thr Ile 115 120 125Glu Leu Leu Glu Gln Leu Thr Arg
Leu Leu Arg Arg Asp Ile Leu Pro 130 135 140Ala Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Val Val Cys Gly Glu Gly Glu Val Trp Gln 165 170 175Gly
Gly Lys Ile Val Ala Ala Ala Pro Val Leu Ala Gly Ala Gly Ile 180 185
190Thr Ala Leu Arg Leu Arg Pro Lys Glu Ala Leu Ala Ile Met Asn Gly
195 200 205Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Ser Arg
Ala Glu 210 215 220Tyr Leu Ala Arg Leu Ala Ala Arg Ile Thr Ala Leu
Ala Ser Phe Ala225 230 235 240Leu Asp Gly Asn Ala Glu His Phe Asp
Ala Gly Leu Phe Ala Leu Lys 245 250 255Pro His Pro Gly Gln Gln Arg
Val Ala Ala Trp Leu Arg Ala Asp Leu 260 265 270Pro Asp Gly Gly Ser
Ala Leu Asn Gly Lys Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Val Asp Ala Leu 290 295 300Pro
Trp Leu Arg Gln Ser Ile Glu Asn Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Val Val Asp Gly Ala Gly Glu Arg Val Leu His Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ala Arg Tyr Ser Asn Gly Leu Pro
Ala Asn Leu Ser Gly Val Glu 370 375 380Gly Pro Arg Ala Ala Ile Asn
His Gly Leu Lys Ala Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Cys Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Ala Val Ala Leu Leu Ile Thr Thr Arg Gln Ala Val
Trp Trp 450 455 460Arg Arg Arg Leu Ser Pro Ser Leu Thr Leu Ser Ala
Pro Leu Arg Glu465 470 475 480Met Ile Asp Ala Leu Gly Ala Asp Ile
Ala Val Leu Glu Glu Asp Arg 485 490 495Arg Leu Glu Pro Asp Leu Arg
Arg Leu Leu Glu Arg Leu Asn Asp Arg 500 505 510Ala Trp Arg Leu Tyr
Glu 515271539DNAUnknownObtained from environmental sample
27atgcatccgc atgagcctgt caccttcggc gaatcgccgc tggcaatcga agatgtcctg
60gccctggccg agcgccgtgc cccggtgcag ctgcaggacg atgccgccta ccgcgagcgc
120atcgcccgcg gcgcgcgctt tctcgacacc ttgctcagca aggaaggggt
gatctacggc 180gtggccaccg gctacggtga ctcctgcgtg gtagcagtgc
cgctggagca tgtggaggcc 240ctgccacagc acctgtatac cttccacggc
tgcggcctgg gcaagctgct cgacgccacc 300gccacccgag ccgtgctggc
cgcgcgcctg cgctcactga gccatggcgt atccggcgtg 360cgtctggagc
tgctggagcg tctgcgcgac ttcctggtac atgacatcct gccgctgatc
420cccgaggaag gctcggtggg tgccagtggc gatttgacac cgctgtccta
tgtcgccgcg 480accttgtccg gcgaacgtga agtgatgtac cagggccaac
gccgtagcag tgccgacgtc 540caccgcgaga tgggctggca gccgctggtg
ctgcggccca aggaggcctt ggcactgatg 600aatggcactg cggtaatgac
cggcctggcc tgcctggcat acgcgcgcgc cgattacctg 660ctgcagttgg
ctacgcgcat caccgccctc aacgtcatcg gcctgcaagg taacccagag
720cacttcgatg agcgcctgtt cgccgccaag cctcatcccg gccagaccca
ggtcgccgcc 780tggttgcgcc aggacctggc catcgacgcg ccactgccac
cgctgcaccg cctgcaggac 840cgctactcgc tgcgttgcgc gccacatgtg
ctgggggtgc tggccgatag cctgggctgg 900ttgcgtggct tcatcgaaac
cgagctcaac agtgccaacg acaatccgat catcgatggc 960gacgccgagc
gcgtgctgca tggcgggcat ttctatggag ggcatatcgc cttcgccatg
1020gacagcctga agaccttggt ggccaatgtc gccgacctgc tcgaccgcca
gctcgcgctg 1080ctggtggacg tgcgctacaa ccatggcctg ccgagcaacc
tgtccggcgc cagcgccgag 1140cggtcgatga tcaaccacgg cttcaaggcc
gtgcagatcg gcgccagcgc ctggaccgcc 1200gaagccctca agcagaccat
gccggccagc gtattttcgc gctccaccga atgccacaac 1260caggacaagg
tgagcatggg caccatcgct gcacgcgatg ccttgcgcgt gctggaactg
1320accgagcagg tcgccgcggc ttcgctgctg gcggctaatc agggtgtctg
gctgcgcgct 1380cgacaggccg acgcacgggc cctgccgccg gcattggcgc
gcatgcacga agcactgctg 1440gaggacttcg ccccggtcat cgaggaccgc
accctggaaa gcgacttgcg cctatgtctg 1500caacgcatta gtcaacgcca
ctggaggctg catgcgtaa 153928512PRTUnknownObtained from environmental
sample 28Met His Pro His Glu Pro Val Thr Phe Gly Glu Ser Pro Leu
Ala Ile1 5 10 15Glu Asp Val Leu Ala Leu Ala Glu Arg Arg Ala Pro Val
Gln Leu Gln 20 25 30Asp Asp Ala Ala Tyr Arg Glu Arg Ile Ala Arg Gly
Ala Arg Phe Leu 35 40 45Asp Thr Leu Leu Ser Lys Glu Gly Val Ile Tyr
Gly Val Ala Thr Gly 50 55 60Tyr Gly Asp Ser Cys Val Val Ala Val Pro
Leu Glu His Val Glu Ala65 70 75 80Leu Pro Gln His Leu Tyr Thr Phe
His Gly Cys Gly Leu Gly Lys Leu 85 90 95Leu Asp Ala Thr Ala Thr Arg
Ala Val Leu Ala Ala Arg Leu Arg Ser 100 105 110Leu Ser His Gly Val
Ser Gly Val Arg Leu Glu Leu Leu Glu Arg Leu 115 120 125Arg Asp Phe
Leu Val His Asp Ile Leu Pro Leu Ile Pro Glu Glu Gly 130 135 140Ser
Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser Tyr Val Ala Ala145 150
155 160Thr Leu Ser Gly Glu Arg Glu Val Met Tyr Gln Gly Gln Arg Arg
Ser 165 170 175Ser Ala Asp Val His Arg Glu Met Gly Trp Gln Pro Leu
Val Leu Arg 180 185 190Pro Lys Glu Ala Leu Ala Leu Met Asn Gly Thr
Ala Val Met Thr Gly 195 200 205Leu Ala Cys Leu Ala Tyr Ala Arg Ala
Asp Tyr Leu Leu Gln Leu Ala 210 215 220Thr Arg Ile Thr Ala Leu Asn
Val Ile Gly Leu Gln Gly Asn Pro Glu225 230 235 240His Phe Asp Glu
Arg Leu Phe Ala Ala Lys Pro His Pro Gly Gln Thr 245 250 255Gln Val
Ala Ala Trp Leu Arg Gln Asp Leu Ala Ile Asp Ala Pro Leu 260 265
270Pro Pro Leu His Arg Leu Gln Asp Arg Tyr Ser Leu Arg Cys Ala Pro
275 280 285His Val Leu Gly Val Leu Ala Asp Ser Leu Gly Trp Leu Arg
Gly Phe 290 295 300Ile Glu Thr Glu Leu Asn Ser Ala Asn Asp Asn Pro
Ile Ile Asp Gly305 310 315 320Asp Ala Glu Arg Val Leu His Gly Gly
His Phe Tyr Gly Gly His Ile 325 330 335Ala Phe Ala Met Asp Ser Leu
Lys Thr Leu Val Ala Asn Val Ala Asp 340 345 350Leu Leu Asp Arg Gln
Leu Ala Leu Leu Val Asp Val Arg Tyr Asn His 355 360 365Gly Leu Pro
Ser Asn Leu Ser Gly Ala Ser Ala Glu Arg Ser Met Ile 370 375 380Asn
His Gly Phe Lys Ala Val Gln Ile Gly Ala Ser Ala Trp Thr Ala385 390
395 400Glu Ala Leu Lys Gln Thr Met Pro Ala Ser Val Phe Ser Arg Ser
Thr 405 410 415Glu Cys His Asn Gln Asp Lys Val Ser Met Gly Thr Ile
Ala Ala Arg 420 425 430Asp Ala Leu Arg Val Leu Glu Leu Thr Glu Gln
Val Ala Ala Ala Ser 435 440 445Leu Leu Ala Ala Asn Gln Gly Val Trp
Leu Arg Ala Arg Gln Ala Asp 450 455 460Ala Arg Ala Leu Pro Pro Ala
Leu Ala Arg Met His Glu Ala Leu Leu465 470 475 480Glu Asp Phe Ala
Pro Val Ile Glu Asp Arg Thr Leu Glu Ser Asp Leu 485 490 495Arg Leu
Cys Leu Gln Arg Ile Ser Gln Arg His Trp Arg Leu His Ala 500 505
510291554DNAUnknownObtained from environmental sample 29atgaaccccg
caacgcctaa ccccgcgacg ctgaatttcg gtgatgcgcc ccttcgcatc 60gaggacgtgg
tggcgctgtc gcgccgccgg ctggacgcac gcgtgtccga ggatcccgcc
120ttccgcgcgc gcatcgcacg cggcgcggat ttcctggatc gcctgctcgc
cgaggaaggg 180gtgatctatg gcgtgaccac gggatacggc gactcgtgca
ccgtcaacat tccaccggcg 240ctggtcgcgg agttgccaca tcatctgttc
gcctaccatg gctgcggtct gggtcgcttc 300ctcgacgaag ccgagacgcg
cgcggtgctg tccgcgcgcc tggcgtcgct ggtgcgtggc 360atgtcgggcg
tcagcctgac actgctcgaa ggcctgggca tgctgctccg gcacgacgtc
420ctgccgctca tcccggcgga ggggtcggtg ggtgccagcg gtgatctcac
gccgctgtcg 480tacgtggcgg cggtcctgtg cggcgagcgc gaagtcatgc
atgcggggcg ccggcgtcct 540gcgggcgagg cattggccga gatcgggatg
acgccgctga aactgcggcc caaggaaggc 600ctggccatca tgaacggcac
cgcggtgatg accgcgctgg cctgcctggc gtaccggcgc 660gcggaatacc
tttcacggct cgccacacgc ctgaccgcgt tcaacgtgct ggccagcgtc
720ggcaacgcgc atcatttcga tgccgtgctg ttcgccgcca agccgcaccc
gggccagtcg 780cgggtggccc agtggttgcg cgaagacctg cacagcgacc
gcccgccgcg caacgaacag 840cgtctccagg accggtattc gctgcgttgc
gcgccgcacg tcatcggcgt gctggaggac 900gcgctcccct tcatgcgcca
actgatcgaa accgaactca acagcgccaa cgacaatccg 960ttgatcgacc
cggaccgtga gcagatcctg cacggcgggc atttctacgg tggccacatc
1020gcattcgcca tggacgcgat gaagaatgcc atcgccaacg tcgccgacgt
gctggaccgc 1080cagctggcct tgctggtgga tgcccgctac aaccatggcc
tgcctgccaa cctgtccggc 1140gcgcagggcg accgtgccgc catcaaccat
ggcttgaagg cgctgcagat cagcgtgtcg 1200gcgtggaccg cggaggcgtt
gaaactgacc atgccggcgt cggtgttctc gcgctccacc 1260gaatgccaca
accaggacaa ggtcagcatg ggtaccatcg cggcccgcga ctgcctgcgc
1320gtgatcgaac tgaccgaaca ggtggtcgcc ggcatgctga tcgcggcccg
ccagggcctt 1380gcgctgaggc tgcaggccgg tgcgcaagcc gacctgcgcg
atggcctggc ggcgatgtac 1440acggacctgt gcgagcgcat accgctggtg
caggaggatc gccccttgga tggggaactg 1500cggcagttgc tggtcgacat
ccgggaagag cgctggcttg tctatgtcga ctga 155430517PRTUnknownObtained
from environmental sample 30Met Asn Pro Ala Thr Pro Asn Pro Ala Thr
Leu Asn Phe Gly Asp Ala1 5 10 15Pro Leu Arg Ile Glu Asp Val Val Ala
Leu Ser Arg Arg Arg Leu Asp 20 25 30Ala Arg Val Ser Glu Asp Pro Ala
Phe Arg Ala Arg Ile Ala Arg Gly 35 40 45Ala Asp Phe Leu Asp Arg Leu
Leu Ala Glu Glu Gly Val Ile Tyr Gly 50 55 60Val Thr Thr Gly Tyr Gly
Asp Ser Cys Thr Val Asn Ile Pro Pro Ala65 70 75 80Leu Val Ala Glu
Leu Pro His His Leu Phe Ala Tyr His Gly Cys Gly 85 90 95Leu Gly Arg
Phe Leu Asp Glu Ala Glu Thr Arg Ala Val Leu Ser Ala 100 105 110Arg
Leu Ala Ser Leu Val Arg Gly Met Ser Gly Val Ser Leu Thr Leu 115 120
125Leu Glu Gly Leu Gly Met Leu Leu Arg His Asp Val Leu Pro Leu Ile
130 135 140Pro Ala Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro
Leu Ser145 150 155 160Tyr Val Ala Ala Val Leu Cys Gly Glu Arg Glu
Val Met His Ala Gly 165 170 175Arg Arg Arg Pro Ala Gly Glu Ala Leu
Ala Glu Ile Gly Met Thr Pro 180 185 190Leu Lys Leu Arg Pro Lys Glu
Gly Leu Ala Ile Met Asn Gly Thr Ala 195 200 205Val Met Thr Ala Leu
Ala Cys Leu Ala Tyr Arg Arg Ala Glu Tyr Leu 210 215 220Ser Arg Leu
Ala Thr Arg Leu Thr Ala Phe Asn Val Leu Ala Ser Val225 230 235
240Gly Asn Ala His His Phe Asp Ala Val Leu Phe Ala Ala Lys Pro His
245 250 255Pro Gly Gln Ser Arg Val Ala Gln Trp Leu Arg Glu Asp Leu
His Ser 260 265 270Asp Arg Pro Pro Arg Asn Glu Gln Arg Leu Gln Asp
Arg Tyr Ser Leu 275 280 285Arg Cys Ala Pro His Val Ile Gly Val Leu
Glu Asp Ala Leu Pro Phe 290 295 300Met Arg Gln Leu Ile Glu Thr Glu
Leu Asn Ser Ala Asn Asp Asn Pro305 310 315 320Leu Ile Asp Pro Asp
Arg Glu Gln Ile Leu His Gly Gly His Phe Tyr 325 330 335Gly Gly His
Ile Ala Phe Ala Met Asp Ala Met Lys Asn Ala Ile Ala 340 345 350Asn
Val Ala Asp Val Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Ala 355 360
365Arg Tyr Asn His Gly Leu Pro Ala Asn Leu Ser Gly Ala Gln Gly Asp
370 375 380Arg Ala Ala Ile Asn His Gly Leu Lys Ala Leu Gln Ile Ser
Val Ser385 390 395 400Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met
Pro Ala Ser Val Phe 405 410 415Ser Arg Ser Thr Glu Cys His Asn Gln
Asp Lys Val Ser Met Gly Thr 420 425 430Ile Ala Ala Arg Asp Cys Leu
Arg Val Ile Glu Leu Thr Glu Gln Val 435 440 445Val Ala Gly Met Leu
Ile Ala Ala Arg Gln Gly Leu Ala Leu Arg Leu 450 455 460Gln Ala Gly
Ala Gln Ala Asp Leu Arg Asp Gly Leu Ala Ala Met Tyr465 470 475
480Thr Asp Leu Cys Glu Arg Ile Pro Leu Val Gln Glu Asp Arg Pro Leu
485 490 495Asp Gly Glu Leu Arg Gln Leu Leu Val Asp Ile Arg Glu Glu
Arg Trp 500 505 510Leu Val Tyr Val Asp 515311554DNAUnknownObtained
from environmental sample 31atgaaccccg caacgcctaa ccccgcgacg
ctgaatttcg gtgatgcgcc tcttcgcatc 60gaggacgtgg tggcgctgtc gcgccgccgg
ctggacgcac gcgtgtccga ggatcccgcc 120ttccgcgcgc gcatcgcacg
cggcgcggat ttcctggatc gcctgctcgc cgaggaaggg 180gtgatctatg
gcgtgaccac gggatacggt gactcgtgca ccgtcaacat tccaccggcg
240ctggtcgcgg agttgccaca tcatctgttc gcctaccatg gctgcggtct
gggtcgcttc 300ctcgacgaag ccgagacgcg cgcggtgctg tccgcgcgcc
tggcgtcgct ggtgcgtggc 360atgtcgggcg tcagcctgac actgctcgaa
ggcctgggca tgctgctccg gcacgacgtc 420ctgccgctca tcccggcgga
ggggtcggtg ggtgccagcg gtgatctcac gccgctgtcg 480tacgtggcgg
cggtcctgtg cggcgagcgc gaagtcatgc atgcggggcg ccggcgtcct
540gcgggcgagg cattggccga gatcgggatg acgccgctga aactgcggcc
caaggaaggc 600ctggccatca tgaacggcac cgcggtgatg accgcgctgg
cctgcctggc gtaccggcgc 660gcggaatacc tttcacggct cgccacacgc
ctgaccgcgt tcaacgtgct ggccagcgcc 720ggcaacgcgc atcatttcga
tgccgtgctg ttcgccgcca agccgcaccc gggccagtcg 780cgggtggccc
agtggttgcg cgaagacctg cacagcgacc gcccgccgcg caacgaacag
840cgtctccagg accggtattc gctgcgttgc gcgccgcacg tcatcggcgt
gctggaggac 900gcgctcccct tcatgcgcca actgatcgaa accgaactca
acagcgccaa cgacaatccg 960ttgatcgacc cggaccgcga gcagatcctg
cacggcgggc atttctacgg tggccacatc 1020gcattcgcca tggacgcgat
gaagaatgcc atcgccaacg tcgccgacgt gctggaccgc 1080cagttggccc
tgctggtgga tgcccgctac aaccatggcc tgcctgccaa cctgtccggc
1140gcgcagggcg accgtgccgc catcaaccat ggcttgaagg cgctgcagat
cagcgtgtcg 1200gcgtggaccg cggaggcgtt gaaactgacc atgccggcgt
cggtgttctc gcgctccacc 1260gagtgccaca accaggacaa ggtcagcatg
ggcaccatcg cggcccgcga ctgcctgcgc 1320gtcatcgagc tgaccgaaca
ggtggtcgcc ggcatgctga tcgcggcccg ccagggcctt 1380gcgctgaggc
tgcaggccgg tgcgcaggcc gacctgcgcg atggcctggc ggcgatgtac
1440gcggacctgt gcgagcgcat accgctggtg caggaggatc gccccctgga
tggggaactg 1500cggcagttgc tggccgacat ccgggaagag cgctggcttg
tctatgtcga ctga 155432517PRTUnknownObtained from environmental
sample 32Met Asn Pro Ala Thr Pro Asn Pro Ala Thr Leu Asn Phe Gly
Asp Ala1 5 10 15Pro Leu Arg Ile Glu Asp Val Val Ala Leu Ser Arg Arg
Arg Leu Asp 20 25 30Ala Arg Val Ser Glu Asp Pro Ala Phe Arg Ala Arg
Ile Ala Arg Gly 35 40 45Ala Asp Phe Leu Asp Arg Leu Leu Ala Glu Glu
Gly Val Ile Tyr Gly 50 55 60Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val Asn Ile Pro Pro Ala65 70 75 80Leu Val Ala Glu Leu Pro His His
Leu Phe Ala Tyr His Gly Cys Gly 85 90 95Leu Gly Arg Phe Leu Asp Glu
Ala Glu Thr Arg Ala Val Leu Ser Ala 100 105 110Arg Leu Ala Ser Leu
Val Arg Gly Met Ser Gly Val Ser Leu Thr Leu 115 120 125Leu Glu Gly
Leu Gly Met Leu Leu Arg His Asp Val Leu Pro Leu Ile 130 135 140Pro
Ala Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser145 150
155 160Tyr Val Ala Ala Val Leu Cys Gly Glu Arg Glu Val Met His Ala
Gly 165 170 175Arg Arg Arg Pro Ala Gly Glu Ala Leu Ala Glu Ile Gly
Met Thr Pro 180 185 190Leu Lys Leu Arg Pro Lys Glu Gly Leu Ala Ile
Met Asn Gly Thr Ala 195 200 205Val Met Thr Ala Leu Ala Cys Leu Ala
Tyr Arg Arg Ala Glu Tyr Leu 210 215 220Ser Arg Leu Ala Thr
Arg Leu Thr Ala Phe Asn Val Leu Ala Ser Ala225 230 235 240Gly Asn
Ala His His Phe Asp Ala Val Leu Phe Ala Ala Lys Pro His 245 250
255Pro Gly Gln Ser Arg Val Ala Gln Trp Leu Arg Glu Asp Leu His Ser
260 265 270Asp Arg Pro Pro Arg Asn Glu Gln Arg Leu Gln Asp Arg Tyr
Ser Leu 275 280 285Arg Cys Ala Pro His Val Ile Gly Val Leu Glu Asp
Ala Leu Pro Phe 290 295 300Met Arg Gln Leu Ile Glu Thr Glu Leu Asn
Ser Ala Asn Asp Asn Pro305 310 315 320Leu Ile Asp Pro Asp Arg Glu
Gln Ile Leu His Gly Gly His Phe Tyr 325 330 335Gly Gly His Ile Ala
Phe Ala Met Asp Ala Met Lys Asn Ala Ile Ala 340 345 350Asn Val Ala
Asp Val Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Ala 355 360 365Arg
Tyr Asn His Gly Leu Pro Ala Asn Leu Ser Gly Ala Gln Gly Asp 370 375
380Arg Ala Ala Ile Asn His Gly Leu Lys Ala Leu Gln Ile Ser Val
Ser385 390 395 400Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro
Ala Ser Val Phe 405 410 415Ser Arg Ser Thr Glu Cys His Asn Gln Asp
Lys Val Ser Met Gly Thr 420 425 430Ile Ala Ala Arg Asp Cys Leu Arg
Val Ile Glu Leu Thr Glu Gln Val 435 440 445Val Ala Gly Met Leu Ile
Ala Ala Arg Gln Gly Leu Ala Leu Arg Leu 450 455 460Gln Ala Gly Ala
Gln Ala Asp Leu Arg Asp Gly Leu Ala Ala Met Tyr465 470 475 480Ala
Asp Leu Cys Glu Arg Ile Pro Leu Val Gln Glu Asp Arg Pro Leu 485 490
495Asp Gly Glu Leu Arg Gln Leu Leu Ala Asp Ile Arg Glu Glu Arg Trp
500 505 510Leu Val Tyr Val Asp 515331557DNAUnknownObtained from
environmental sample 33gtggggcagt tggcggcgac aaagacaatt gaaatcggcg
ggcatgacct gacgattgag 60caggtgtggc aattggctca tgccgtggcc gagccgcggc
tgagcgagct agccctcgat 120cgcgtccaac gctcggcccg tcatgtggag
acggcgctgg cgcgtgatgg caccatctat 180ggcgtcacca cgggctatgg
cgacagctgc accacaaacg tgccaaccgc tctggttgag 240caactaccgc
ttcagctcac gcgctatcat ggctgcggta tggggcgcat actgacgcct
300gaagagaccc gggcggtaat ggctgttcgg ccggccgcgc tcgtgcgcgg
ctggtctggg 360gtttcgcccg agcttgtgac gctgctaacg gcgatgcttc
ccgatatttt gccccaaatt 420ccagcagagg gatctgtggg cgcgagtgga
gacttgaccc cgctgagcta tttggcgggc 480gcgctggctg gcgagcgaga
cgtgctgtac gggcgccgta aaatgaccgc cgccgccgcg 540ttcgctgaaa
ctgggctcca accgatcgtc ttgcgcccca aagaggcgct ggcgatcatg
600aacggcacgg ccgtgatgac gggcctcgct gtgcttgcgt tcgttcgcgc
cgaaggcctg 660gcgcgcctga cttccagact atcagcgatg gcttcgctgg
ccctgctggg caatccgagc 720catttcgacg cccgcctgtc gaaggcgaag
ccgcatgccg ggcaaggccg ggtcgcggcg 780cgtattgcgg cggatctcgc
gccgtttgta cggcaaggcg cgccgtcgcg attgcaggat 840cgctattcga
tacgttgcgc gccccacgtc attggcgtgg ttgaagacat gctggcgccg
900ttcaggtcga tgattgagac ggagatcaac agcgccaacg acaatccgtt
gtttgatccc 960gacaccggtg agacgttgca tggcgggcat ttctacggcg
ggcatatcgc gtttgcgatg 1020gacggcctca agacgctggt cgccaacctg
gctgatttga tggatcgtca gtttgcgctg 1080ctggcggata cgcgtttcag
caacggcttg ccgccgaatc tgtcaggggc gaccggacag 1140cgcgcttcga
tcaatcatgg cttgaaggca gtgcagatcg cggtgtccgc ttggacagca
1200gaggcgctca agggcacgat gccggcgagc gtgttctcac gctcgactga
gtgtcacaat 1260caggacaagg tctcgatggg cacgattgct gcgcgggacg
ccctgcgtgt ccttgagctg 1320acagagcagg tcgcggctgc tcatatcatt
gcttgcgccc agggcctgcg cctgcggctg 1380cgcgccggcg aaataacgcc
cgccatgatt gggcctggca tgacggcgct gctggagctt 1440tcgccgctca
ttgaagaaga cgcgcccctc gatacacggt tgaccgaact tacgggcgcc
1500ataagtcggc gcgaatttcg ccttgattgg ccggaggatg cggagcggat cgaatga
155734518PRTUnknownObtained from environmental sample 34Met Gly Gln
Leu Ala Ala Thr Lys Thr Ile Glu Ile Gly Gly His Asp1 5 10 15Leu Thr
Ile Glu Gln Val Trp Gln Leu Ala His Ala Val Ala Glu Pro 20 25 30Arg
Leu Ser Glu Leu Ala Leu Asp Arg Val Gln Arg Ser Ala Arg His 35 40
45Val Glu Thr Ala Leu Ala Arg Asp Gly Thr Ile Tyr Gly Val Thr Thr
50 55 60Gly Tyr Gly Asp Ser Cys Thr Thr Asn Val Pro Thr Ala Leu Val
Glu65 70 75 80Gln Leu Pro Leu Gln Leu Thr Arg Tyr His Gly Cys Gly
Met Gly Arg 85 90 95Ile Leu Thr Pro Glu Glu Thr Arg Ala Val Met Ala
Val Arg Pro Ala 100 105 110Ala Leu Val Arg Gly Trp Ser Gly Val Ser
Pro Glu Leu Val Thr Leu 115 120 125Leu Thr Ala Met Leu Pro Asp Ile
Leu Pro Gln Ile Pro Ala Glu Gly 130 135 140Ser Val Gly Ala Ser Gly
Asp Leu Thr Pro Leu Ser Tyr Leu Ala Gly145 150 155 160Ala Leu Ala
Gly Glu Arg Asp Val Leu Tyr Gly Arg Arg Lys Met Thr 165 170 175Ala
Ala Ala Ala Phe Ala Glu Thr Gly Leu Gln Pro Ile Val Leu Arg 180 185
190Pro Lys Glu Ala Leu Ala Ile Met Asn Gly Thr Ala Val Met Thr Gly
195 200 205Leu Ala Val Leu Ala Phe Val Arg Ala Glu Gly Leu Ala Arg
Leu Thr 210 215 220Ser Arg Leu Ser Ala Met Ala Ser Leu Ala Leu Leu
Gly Asn Pro Ser225 230 235 240His Phe Asp Ala Arg Leu Ser Lys Ala
Lys Pro His Ala Gly Gln Gly 245 250 255Arg Val Ala Ala Arg Ile Ala
Ala Asp Leu Ala Pro Phe Val Arg Gln 260 265 270Gly Ala Pro Ser Arg
Leu Gln Asp Arg Tyr Ser Ile Arg Cys Ala Pro 275 280 285His Val Ile
Gly Val Val Glu Asp Met Leu Ala Pro Phe Arg Ser Met 290 295 300Ile
Glu Thr Glu Ile Asn Ser Ala Asn Asp Asn Pro Leu Phe Asp Pro305 310
315 320Asp Thr Gly Glu Thr Leu His Gly Gly His Phe Tyr Gly Gly His
Ile 325 330 335Ala Phe Ala Met Asp Gly Leu Lys Thr Leu Val Ala Asn
Leu Ala Asp 340 345 350Leu Met Asp Arg Gln Phe Ala Leu Leu Ala Asp
Thr Arg Phe Ser Asn 355 360 365Gly Leu Pro Pro Asn Leu Ser Gly Ala
Thr Gly Gln Arg Ala Ser Ile 370 375 380Asn His Gly Leu Lys Ala Val
Gln Ile Ala Val Ser Ala Trp Thr Ala385 390 395 400Glu Ala Leu Lys
Gly Thr Met Pro Ala Ser Val Phe Ser Arg Ser Thr 405 410 415Glu Cys
His Asn Gln Asp Lys Val Ser Met Gly Thr Ile Ala Ala Arg 420 425
430Asp Ala Leu Arg Val Leu Glu Leu Thr Glu Gln Val Ala Ala Ala His
435 440 445Ile Ile Ala Cys Ala Gln Gly Leu Arg Leu Arg Leu Arg Ala
Gly Glu 450 455 460Ile Thr Pro Ala Met Ile Gly Pro Gly Met Thr Ala
Leu Leu Glu Leu465 470 475 480Ser Pro Leu Ile Glu Glu Asp Ala Pro
Leu Asp Thr Arg Leu Thr Glu 485 490 495Leu Thr Gly Ala Ile Ser Arg
Arg Glu Phe Arg Leu Asp Trp Pro Glu 500 505 510Asp Ala Glu Arg Ile
Glu 515351563DNAUnknownObtained from environmental sample
35atgcatcccg ctgaccagaa gaacgcaacc acaacccgta caattaaact cgacaatggt
60cggctgagaa tcgaggacat tatcgatatc gcggaggggt cggcccgtgt cgtattgtcc
120gattcgcatg aattctgcgc cgccattgac cgaggggcgg attttctcga
ccgtttgctg 180cgtgaagagg gtacgatcta tggtgtcacc accggctacg
gcgattcgtg caccgtgacg 240gtcccggttg agctgatcgg cgagctgccg
cgccatctct acacctatca cggctgtggg 300ctgggcgact atttgagtcc
ggtacagaca cgcgcagtga tggcgactcg cctcacatcg 360ttgtgcaaag
ggttctccgg cgtcagtctg gaactgctgc ggcaaatcgt aaagttactc
420cagtgcgatc tattaccatt gattccatcc gaaggctcag tgggtgccag
cggtgatctc 480actccgttat cgtaccttgc tgccgttttg tgcggcgagg
gtgaagtttg gcgcaacggc 540gcccgtttaa gcgccgctca ggcactgact
gaggcaggga tgacaccgct gcggttgagg 600ccaaaagaag gactggcgat
catgaacggg acgtcggtaa tgaccggttt ggcttgcctt 660gcgtatgcac
gtgccgatta tctcacccgg ctggtcactc gcatcaccgc cctagcttcc
720tttgcgcttg acggcaacgc gcagcatttt gatcctaagt tattctcggt
gaagccgcat 780ccaggcctgc agcgcgtggc gggctggttg cgtcaagact
tgccgtgcga tcggcgtgac 840cgcgacggaa agcgtctaca ggaccgttat
tcggtgcgtt gcgcgcccca cgtgatcggt 900gttctagcag acgccacgcc
gtggctgcgc gactccatcg aaaatgaact caacagcgcc 960aatgacaatc
ccatcattga tgcggaaggc gaaggcgtgc tgtatggtgg ccacttttac
1020ggtgggcata tcgcgtttgc catggacagc atgaaaaatg ccgtcgcaaa
cctggcggat 1080ctgcttgacc ggcagatggc cctgctggtc gacagccgct
acaacaatgg tttggcgccc 1140aacttgtctg gagctcatgg gccacgtgca
gcgatcaatc atgggctcaa gggcctgcag 1200attagtgctt cggcatgggc
agcggaagcg ctcaagttaa cgatgccggc gtcggtgttc 1260tcgcgttcga
cggagtgcca caatcaggat aaagtcagca tgggcaccat tgcggcacgg
1320gattctctgc gggtgctcga gctaaccgag caggtcgctg ccgcgatgct
gatagccgtg 1380cgacaaggtg tctggctgcg ttgccggctg aaccgatcca
tggctccgga ggcgacgctg 1440cggaacatga tggacgcgct aggcgctgat
atcccggtaa tcgaggagga ccgcaagcta 1500gagccggatc tcagactaat
gctggagcgc attcgcggtc gggcctggaa gctatatgaa 1560taa
156336520PRTUnknownObtained from environmental sample 36Met His Pro
Ala Asp Gln Lys Asn Ala Thr Thr Thr Arg Thr Ile Lys1 5 10 15Leu Asp
Asn Gly Arg Leu Arg Ile Glu Asp Ile Ile Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Val Leu Ser Asp Ser His Glu Phe Cys Ala Ala 35 40
45Ile Asp Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Val Glu Leu Ile Gly Glu Leu Pro Arg His Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Asp Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Arg Gln Ile
Val Lys Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Ala Arg Leu Ser Ala Ala Gln Ala Leu Thr Glu Ala 180 185
190Gly Met Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ser Val Met Thr Gly Leu Ala Cys Leu Ala Tyr
Ala Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala Gln His
Phe Asp Pro Lys Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Arg Arg Asp Arg Asp Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Thr Pro Trp Leu Arg Asp Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Gly Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Ala Pro Asn Leu Ser Gly 370 375 380Ala His Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Ala Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Met Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Met Ala Pro
Glu Ala Thr Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Gly Ala
Asp Ile Pro Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Met Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys
Leu Tyr Glu 515 520371566DNAUnknownObtained from environmental
sample 37atgcatcccg ttgacttgaa gaagaactca aactcagtcc gtacgattca
cttcgacgat 60ggacggctga aagtcgagga tattgtcgat atcgcggaag ggtcggctag
ggtagcactg 120tccgatgctt ctgaattccg ctccgcgatt gcgcaagggg
cggactttct ggaccgtttg 180ctgcgcgaag agggcacgat ttatggcgtc
actaccggct acggcgattc atgcaccgtg 240acagtcccgc tggatttggt
cgccgagttg ccgcgccatc tctacactta tcatggttgc 300gggctagggg
agtatttaag tcccgtacag acgcgggccg tgatcgccac ccgcctcacg
360tcgttgtgca aaggattctc tggcgtcagt ctcgaactgc tgcatcaact
tgtgaggttg 420ctgcagtgcg atttattgcc attgattcca tctgaaggtt
cggtgggcgc cagcggcgat 480ctcaccccac tatcttatct cgctgccgtg
ttgtgcggcg agggcgaagt ttggcgcgaa 540ggcgtccata cgagcgccgc
gcaggcactg gctgaggccg gtatcgcacc gctacggttg 600aggccaaaag
aaggactggc gatcatgaat gggacgtcgg tcatgaccgc tttggcgtgc
660cttgcttatg cgcgcgccga ttacctcacc cggctagtca ctcgcatcac
cgcattgacg 720tcgtttgcgc tggacggcaa tgcgcaccat ttcgaggcta
cgttgttctc agtgaagccg 780cacccaggcc agcagcgggt ggccggttgg
ttgcgtcaag acttggcgtg cgatcagctg 840gaccccaacg gaaagcgtct
ccaggatcgc tactcgatcc gttgcgcgcc ccacgtgatc 900ggtgttctcg
ccgatgcgct gccatggttg cgcgagtcca tcgaaaatga actcaacagc
960gccaacgata atccgatcat cgacgcggag ggcgaacggg tgctctatgg
cggtcacttc 1020tacggggggc atattggatt tgccatggac agcatgaaaa
atgccgtcgc aaacctggcg 1080gatctgcttg accggcagat ggccctgctg
gtcgacagcc ggtacaataa tgggttgcca 1140gccaatttgt ctggggttca
agggccacgc gcagcgatca atcatggcct caaggggttg 1200cagatcagtg
tttcggcatg gaccgcggag gcgctcaagt tgaccatgcc ggcgtcggtg
1260ttctcgcgtt cgaccgagtg ccacaaccag gacaaagtca gcatgggtac
gattgcggca 1320cgcgattgtt tgcgtgtgct cgaattgacg gagcaagtcg
ccgcggcgtt gctaatcacc 1380gtgcgacagg gggtctggct ccgttgcagg
ctgaatcgat cagccgctcc cgaggcgacg 1440ttgaagaaaa tgatggacgc
cttgggcgct gatatcgccg agattgagga ggaccgcagg 1500ctagagccgg
atttgcgatt actactggag cgcatccgcg accgtgtctg ggaattgtat 1560gaataa
156638521PRTUnknownObtained from environmental sample 38Met His Pro
Val Asp Leu Lys Lys Asn Ser Asn Ser Val Arg Thr Ile1 5 10 15His Phe
Asp Asp Gly Arg Leu Lys Val Glu Asp Ile Val Asp Ile Ala 20 25 30Glu
Gly Ser Ala Arg Val Ala Leu Ser Asp Ala Ser Glu Phe Arg Ser 35 40
45Ala Ile Ala Gln Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu
50 55 60Gly Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val65 70 75 80Thr Val Pro Leu Asp Leu Val Ala Glu Leu Pro Arg His
Leu Tyr Thr 85 90 95Tyr His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro
Val Gln Thr Arg 100 105 110Ala Val Ile Ala Thr Arg Leu Thr Ser Leu
Cys Lys Gly Phe Ser Gly 115 120 125Val Ser Leu Glu Leu Leu His Gln
Leu Val Arg Leu Leu Gln Cys Asp 130 135 140Leu Leu Pro Leu Ile Pro
Ser Glu Gly Ser Val Gly Ala Ser Gly Asp145 150 155 160Leu Thr Pro
Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu 165 170 175Val
Trp Arg Glu Gly Val His Thr Ser Ala Ala Gln Ala Leu Ala Glu 180 185
190Ala Gly Ile Ala Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile
195 200 205Met Asn Gly Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala
Tyr Ala 210 215 220Arg Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile
Thr Ala Leu Thr225 230 235 240Ser Phe Ala Leu Asp Gly Asn Ala His
His Phe Glu Ala Thr Leu Phe 245 250 255Ser Val Lys Pro His Pro Gly
Gln Gln Arg Val Ala Gly Trp Leu Arg 260 265 270Gln Asp Leu Ala Cys
Asp Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln 275 280 285Asp Arg Tyr
Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala 290 295 300Asp
Ala Leu
Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser305 310 315
320Ala Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Arg Val Leu Tyr
325 330 335Gly Gly His Phe Tyr Gly Gly His Ile Gly Phe Ala Met Asp
Ser Met 340 345 350Lys Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala 355 360 365Leu Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser 370 375 380Gly Val Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu385 390 395 400Gln Ile Ser Val Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met 405 410 415Pro Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys 420 425 430Val
Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu 435 440
445Leu Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Thr Val Arg Gln Gly
450 455 460Val Trp Leu Arg Cys Arg Leu Asn Arg Ser Ala Ala Pro Glu
Ala Thr465 470 475 480Leu Lys Lys Met Met Asp Ala Leu Gly Ala Asp
Ile Ala Glu Ile Glu 485 490 495Glu Asp Arg Arg Leu Glu Pro Asp Leu
Arg Leu Leu Leu Glu Arg Ile 500 505 510Arg Asp Arg Val Trp Glu Leu
Tyr Glu 515 520391563DNAUnknownObtained from environmental sample
39atgcatcccg ctgacctgaa gacctcaatc ttacctcgga cgattgaatt cgaccagcga
60tggctgaaaa tcgaagacat tgtcgatatc gcggaagggt cgactagggt agcattgtcc
120gatactgctg catttcgctc tggcattaag cgaggcgcgg ccttcctgga
ccgtctgctg 180cacgaagagg gcacgatcta tggcgttact accggctacg
gcgattcgtg cacggtgacg 240gtgccgcttg agctagtcgc cgagttgccg
cgccagctct atgtctatca cggctgtggg 300ctgggcgagt atttgagtcc
ggtacagacg cgcgccgtgt tggcaacccg cctcacatcg 360ttgtgcaaag
ggttttctgg cgtgagcttg gaactgctgc aacaaatcgt gaggttactc
420cagtgcgatc tattgccatt gattccgtcg gaaggctcgg tgggtgccag
cggcgatctc 480actccgctat cgtacctcgc tgcggttttg tgcggcgagg
gcgacgtttg gcgtaacggc 540gtccatgtaa gcgccgcgca agcactagcc
gagggcggta tcagaccgct gcgtttgagg 600cccaaagaag gattggcgat
catgaatggg acagcagtca tgaccgcttt ggcttgcctt 660gcttatgtgc
gcgccgatta tcttacccga ctggtgactc ggatcaccgc actggcgtcg
720tttgcgcttg acggcaatgc gcaccatttc gatgccacat tgttcgcagt
gaaaccgcat 780ccgggcatgc agcgagtcgc cggttggttg cgtcaagact
tgccgtgcga tcacctggac 840accaatagaa agcggctgca ggatcgctat
tcgctccgct gcgcgcccca cgtgatcggt 900gttctagccg acgcgctgcc
atggttgcgc gagtctatcg aaaatgaact caacagtgcc 960aacgacaatc
ccatcatcga tgcggaaggc caaggcgtgt tgtctggcgg tcacttttac
1020ggcgggcata tcgcgtttgc catggacagc atgaaaaatg ccgtcgctaa
cctggcggat 1080ctgctcgacc ggcagatggc actgctggtc gatggccgct
acaacaatgg gctgccggcc 1140aacttgtctg gggctcaagg cccgcgcgcc
gcgatcaatc atggactcaa ggggttgcag 1200atcagcgctt cggcgtggac
tgcggaagcg ctcaagttga ccatgccggc ctcggtgttc 1260tcgcgctcga
ccgagtgcca taaccaggac aaagttagtc tgggtacgat tgcagcacgc
1320gattgtttgc gggtgctcga attgacggag caagtcgccg cggcgctgct
cgttgccgta 1380cgacaaggag tctggctccg ttgcaggatg aatcgatccg
tcgctttggg aatggcgcta 1440aggaacatga tggatgccct gagcaccgat
atcaacgcga tagaggaaga tcgtcgactg 1500gaggcggatt tgcgattact
gctggagcgc atccatagcc gtgtctggaa gctgtatgaa 1560taa
156340520PRTUnknownObtained from environmental sample 40Met His Pro
Ala Asp Leu Lys Thr Ser Ile Leu Pro Arg Thr Ile Glu1 5 10 15Phe Asp
Gln Arg Trp Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Thr Arg Val Ala Leu Ser Asp Thr Ala Ala Phe Arg Ser Gly 35 40
45Ile Lys Arg Gly Ala Ala Phe Leu Asp Arg Leu Leu His Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Leu Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Asn Gly Val His Val Ser Ala Ala Gln Ala Leu Ala Glu Gly 180 185
190Gly Ile Arg Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
His Leu Asp Thr Asn Arg Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Leu Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Gln Gly Val Leu Ser
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Gly Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Ala Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Val Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Met Asn Arg Ser Val Ala Leu
Gly Met Ala Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asn Ala Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Ala Asp
Leu Arg Leu Leu Leu Glu Arg Ile His 500 505 510Ser Arg Val Trp Lys
Leu Tyr Glu 515 520411563DNAUnknownObtained from environmental
sample 41atgcatcccg ctgaccagaa gaacgcaacc acagcccgta caattaaact
cgacaatggt 60cggctgagaa tcgaggacat tatcgatatc gcggaagggt cggcccgtgt
cgtattgtcc 120gattcgcatg aattctgcgc cgccattaac cgaggggcgg
attttctcga ccgtttgctg 180cgtgaagagg gtacgatcta tggtgtcacc
accggctacg gcgattcgtg caccgtgacg 240gtcccggttg agctgatcgg
cgagctgccg cgccatctct acacctatca cggctgtggg 300ctgggcgagt
atttgagtcc gatacagaca cgcgcagtga tggcgactcg cctcacatcg
360ttgtgcaaag ggttctccgg cgtcagtctg gaactgctgc ggcaaatcgt
gaagttactc 420cagtgcgatc tattaccatt gattccatcc gaaggctcag
tgggtgccag cggtgatctc 480actccgttat cgtaccttgc tgccgttttg
tgcggcgagg gtgaagtttg gcgcaacggc 540gtccgtttaa gcgccgctca
ggcactgact gaggcaggga tgacaccgct gcggttgagg 600ccaaaagaag
gactggcgat catgaacggg acgtcggtaa tgaccggttt ggcttgcctt
660gcgtatgcac gtgccgatta tctcacccgg ctggtcactc gcatcaccgc
cctagcgtcc 720tttgcgcttg acggcaacgc gcaccatttt gatcctaagt
tattctcggt gaagccgcat 780ccaggcctgc agcgcgtggc gggctggttg
cgtcaagact tgccgtgcga tcagcgtgac 840cgcaacggaa agcgtctaca
ggaccgttat tcggtgcgtt gcgcgcccca cgtgatcggt 900gttctagcag
acgccacgcc gtggctgcgc gactccatcg aaaatgaact caacagcgcc
960aacgacaatc ccatcattga tgcggaaggc gaaggcgtgc tgtatggtgg
ccacttttac 1020ggtgggcata tcgcgtttgc catggacagc atgaaaaatg
ccgtcgcaaa cctggcggat 1080ctgcttgacc ggcagatggc cctgctggtc
gacagccgct acaacaatgg tttggcggcc 1140aacttgtctg gagctcatgg
tccacgtgca accatcaatc atgggctcaa aggcttgcag 1200attagtgctt
cggcatgggc ggcggaagcg ctcaagttaa cgatgccggc gtcggtgttc
1260tcgcgttcga ccgagtgcca caatcaggat aaagtcagca tgggcaccat
tgcggcacgg 1320gattctctgc gggtgctcga gctaaccgag caggtcgctg
ccgcgatgct gatagccgtg 1380cgacaaggtg tctggctgcg ttgccggctg
aaccgatcca tggctccgga ggcgacgctg 1440cggaacatga tggacgcgct
aggcgctgat atcccggtaa tcgaggagga ccgcaagcta 1500gagccggatc
tcagactagt gctggagcgc attcgcggtc gggcctggaa gctatatgaa 1560taa
156342520PRTUnknownObtained from environmental sample 42Met His Pro
Ala Asp Gln Lys Asn Ala Thr Thr Ala Arg Thr Ile Lys1 5 10 15Leu Asp
Asn Gly Arg Leu Arg Ile Glu Asp Ile Ile Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Val Leu Ser Asp Ser His Glu Phe Cys Ala Ala 35 40
45Ile Asn Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Val Glu Leu Ile Gly Glu Leu Pro Arg His Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Ile
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Arg Gln Ile
Val Lys Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val Arg Leu Ser Ala Ala Gln Ala Leu Thr Glu Ala 180 185
190Gly Met Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ser Val Met Thr Gly Leu Ala Cys Leu Ala Tyr
Ala Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Pro Lys Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Gln Arg Asp Arg Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Thr Pro Trp Leu Arg Asp Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Gly Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Ala Ala Asn Leu Ser Gly 370 375 380Ala His Gly Pro Arg Ala Thr
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Ala Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Met Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Met Ala Pro
Glu Ala Thr Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Gly Ala
Asp Ile Pro Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Val Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys
Leu Tyr Glu 515 520431557DNAUnknownObtained from environmental
sample 43atgcaaccag gtgacatgaa cacttcaacc tcgacccgca ggattacgtt
cgacaacgga 60ccggtgagaa tcgaggacat cgtggacatc gcggcagggt ccgcaagcgt
ggcattgtct 120gatgcttccg aatttcgcgc agccattacc cgaggggcgg
actttctgga ctgtctggcg 180cgcgaagagg gcacgatcta tggcgtcacc
accggctacg gcgattcatg caccgtgacg 240gtcccgtttg agttggtcgc
cgagctgcca cgccatctct acacgtatca cggctgcggg 300ctgggggagt
atttaagtcc ggcacagacg cgtgccgtga tcgcaacccg cctcacgtcg
360ctgtctaaag gcttctctgg tgtcagtctg gaactgctgg aacagatggt
gcggctactg 420cagtgcgatt tactgccgtt gattccgtgt gaagggtcgg
tgggtgccag cggcgatcta 480actccgctat cctacctcgc cgctgttttg
tgcggcgagg gtgaagtttg gcgcaacggc 540gtccaggtag gcgccgcgca
ggcactgtct gaggccggca tcacgccgct gcgactccga 600cccaaagaag
gactggccgt catgaacggg accgccgtca tgacggcttt ggcgtgcctc
660gcttacgtgc gcgccgatta tcttacccgg ctggcaacgc gcattaccgc
attggcaccg 720tttgcgctcg acggcaatgc gcaccatttc gatgcgacgc
tattttcagt gaagccgcat 780ccaggccagc aacgcgtggc cggctggctg
cgccgagact tgccgtgcga tcagctggat 840acgaacggaa aacgtctgca
ggaccgctat tcgatacgtt gcgcgccgca cgtgattggt 900gttctcgccg
acgcactgcc ctggttgcgc gagttcatcg aaaatgaact taatagcgcc
960aacgataatc cgatcgttga tgcggagagc aaaagcgtac tgtacggcgg
tcatttttac 1020ggcgggcaca tcgcgtttgc catggacagc atgaaaaatg
ccgtcgccaa cctggcggat 1080ctgatggacc ggcagatggc gctgctggtg
gatacccgct acaacaatgg cttgccggcc 1140aacttgtccg gggttcaagg
gccgcgtgca gcgatcaacc atgggctcaa ggcgttgcag 1200atcagcgcgt
cggcatggac ggcggaagcg ctcaagttga ccatgccggc gtcggtgttt
1260tcgcgctcga cggagtgtca taaccaggac aaggtcagca tgggtacgat
tgcggcacgc 1320gattgtttgc gggtgctcga attggtggag caagtcgccg
cggcgctgct catcgcagtg 1380cggcagggag tctggctccg ttgcaaactg
aatcggtccc cggggacaac cttaaagagc 1440atgatggacg cgctgagcac
tgatatcgac gtgatcgagg aagatcgtcg gcttgagcca 1500gatttgcgac
tactgctgga gcgcatccgc gaccgtgtct ggaaactgta tgaataa
155744518PRTUnknownObtained from environmental sample 44Met Gln Pro
Gly Asp Met Asn Thr Ser Thr Ser Thr Arg Arg Ile Thr1 5 10 15Phe Asp
Asn Gly Pro Val Arg Ile Glu Asp Ile Val Asp Ile Ala Ala 20 25 30Gly
Ser Ala Ser Val Ala Leu Ser Asp Ala Ser Glu Phe Arg Ala Ala 35 40
45Ile Thr Arg Gly Ala Asp Phe Leu Asp Cys Leu Ala Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Phe Glu Leu Val Ala Glu Leu Pro Arg His Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Ala
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Ser
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Glu Gln Met
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Cys
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val Gln Val Gly Ala Ala Gln Ala Leu Ser Glu Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Val Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Ala Thr Arg Ile Thr
Ala Leu Ala Pro225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Gly Trp Leu Arg Arg 260 265 270Asp Leu Pro Cys Asp
Gln Leu Asp Thr Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Phe Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Val Asp Ala Glu Ser Lys Ser Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Met Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Thr Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370
375 380Val Gln Gly Pro Arg Ala Ala Ile Asn His Gly Leu Lys Ala Leu
Gln385 390 395 400Ile Ser Ala Ser Ala Trp Thr Ala Glu Ala Leu Lys
Leu Thr Met Pro 405 410 415Ala Ser Val Phe Ser Arg Ser Thr Glu Cys
His Asn Gln Asp Lys Val 420 425 430Ser Met Gly Thr Ile Ala Ala Arg
Asp Cys Leu Arg Val Leu Glu Leu 435 440 445Val Glu Gln Val Ala Ala
Ala Leu Leu Ile Ala Val Arg Gln Gly Val 450 455 460Trp Leu Arg Cys
Lys Leu Asn Arg Ser Pro Gly Thr Thr Leu Lys Ser465 470 475 480Met
Met Asp Ala Leu Ser Thr Asp Ile Asp Val Ile Glu Glu Asp Arg 485 490
495Arg Leu Glu Pro Asp Leu Arg Leu Leu Leu Glu Arg Ile Arg Asp Arg
500 505 510Val Trp Lys Leu Tyr Glu 515451566DNAUnknownObtained from
environmental sample 45atgcattccg ttgacttgaa gaagacctca acctcagtcc
gtacgattcg attcgacgat 60ggacggctga aaatcgagga cattgtcgac atcgcggaag
ggtcggctag cgtgacatta 120tccgatgctt ctgaattccg ctccgccatt
gcgcgagggg cggactttct ggaccgtctg 180ctgcgcgaag agggcacgat
ttatggtgtc agcaccggct acggcgattc gtgcacggtg 240acggtcccgc
ttgagttggt cgccgagttg ccgcgccatc tctacactta tcacggctgc
300gggctggggg agtatttaac tccggtacag acgcgggccg tgatcgccac
ccgcctcacg 360tcgttgtgca aaggattctc tggcgtcagt ctggaactgc
tgcaacaaat ggtgaggtta 420ctgcagtacg atttattgcc attgattcca
tctgaaggtt cggtgggcgc cagcggcgat 480ctcactccgc tatcctatct
cgctgccgtg ttgtgcggcg agggtgaagt tttgcgcaac 540ggcgtccatg
cgagcgccgc gcaggcactg actgaggccg gtatcgcacc gctgcggttg
600aggccaaaag aaggactggc catcatgaat gggacgtccg tcatgaccgc
tttggcgtgc 660cttgcttatg cgcgcgccga ttacctcacg cggctcgtca
ctcgcatcac cgcattggcg 720tcgtttgcgc tggacggcaa tgcgcaccat
ttcgatgcta cgttattctc agtgaagccg 780catccagggc agcagcgggt
ggccggttgg ttgcgtcaag acttggcttg cgatcagctg 840gaccccaacg
gaaagcgcct ccaggatcgc tactcgatcc gttgcgcgcc ccacgtgatc
900ggtgttctcg ccgatgcgct gccatggttg cgcgagtcca tcgaaaatga
actcaacagc 960gccaacgaca atcccatcat tgatgcggag ggcgaacgcg
tgctctatgg cggtcatttt 1020tacggcgggc atatcgcgtt tgctatggac
agcatgaaaa atgccgtcgc aaacctggcg 1080gatctgcttg accggcagat
ggcactgctg gtcgacagcc gctacaataa cgggttgccc 1140gccaatttgt
ctggggttcg agggccgcgt gcagcgatca atcatgggct caaggggttg
1200cagatcagtg cttcggcatg gaccgcagag gcgctcaagt tgaccatgcc
ggcgtcggtg 1260ttctctcgtt cgaccgagtg ccacaaccag gacaaagtta
gcatgggtac cattgcggca 1320cgcgattgtt tgcgtgtgct cgaattggcg
gagcaagtcg ccgcggcgct gctgatcgct 1380gtgcggcagg gagtctggct
ccgttgcaga ctgaatcgat ccgtggctcc gggggcgacg 1440ttgaagaaca
tgatggacgc cttgggcgct gatatcactg tgatcgagga agatcgtcga
1500ctggagccgg atttgcgatt actgctggag cgtatccgtg gccgcgcctg
gaacctgtat 1560gaataa 156646521PRTUnknownObtained from
environmental sample 46Met His Ser Val Asp Leu Lys Lys Thr Ser Thr
Ser Val Arg Thr Ile1 5 10 15Arg Phe Asp Asp Gly Arg Leu Lys Ile Glu
Asp Ile Val Asp Ile Ala 20 25 30Glu Gly Ser Ala Ser Val Thr Leu Ser
Asp Ala Ser Glu Phe Arg Ser 35 40 45Ala Ile Ala Arg Gly Ala Asp Phe
Leu Asp Arg Leu Leu Arg Glu Glu 50 55 60Gly Thr Ile Tyr Gly Val Ser
Thr Gly Tyr Gly Asp Ser Cys Thr Val65 70 75 80Thr Val Pro Leu Glu
Leu Val Ala Glu Leu Pro Arg His Leu Tyr Thr 85 90 95Tyr His Gly Cys
Gly Leu Gly Glu Tyr Leu Thr Pro Val Gln Thr Arg 100 105 110Ala Val
Ile Ala Thr Arg Leu Thr Ser Leu Cys Lys Gly Phe Ser Gly 115 120
125Val Ser Leu Glu Leu Leu Gln Gln Met Val Arg Leu Leu Gln Tyr Asp
130 135 140Leu Leu Pro Leu Ile Pro Ser Glu Gly Ser Val Gly Ala Ser
Gly Asp145 150 155 160Leu Thr Pro Leu Ser Tyr Leu Ala Ala Val Leu
Cys Gly Glu Gly Glu 165 170 175Val Leu Arg Asn Gly Val His Ala Ser
Ala Ala Gln Ala Leu Thr Glu 180 185 190Ala Gly Ile Ala Pro Leu Arg
Leu Arg Pro Lys Glu Gly Leu Ala Ile 195 200 205Met Asn Gly Thr Ser
Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Ala 210 215 220Arg Ala Asp
Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu Ala225 230 235
240Ser Phe Ala Leu Asp Gly Asn Ala His His Phe Asp Ala Thr Leu Phe
245 250 255Ser Val Lys Pro His Pro Gly Gln Gln Arg Val Ala Gly Trp
Leu Arg 260 265 270Gln Asp Leu Ala Cys Asp Gln Leu Asp Pro Asn Gly
Lys Arg Leu Gln 275 280 285Asp Arg Tyr Ser Ile Arg Cys Ala Pro His
Val Ile Gly Val Leu Ala 290 295 300Asp Ala Leu Pro Trp Leu Arg Glu
Ser Ile Glu Asn Glu Leu Asn Ser305 310 315 320Ala Asn Asp Asn Pro
Ile Ile Asp Ala Glu Gly Glu Arg Val Leu Tyr 325 330 335Gly Gly His
Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Met 340 345 350Lys
Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln Met Ala 355 360
365Leu Leu Val Asp Ser Arg Tyr Asn Asn Gly Leu Pro Ala Asn Leu Ser
370 375 380Gly Val Arg Gly Pro Arg Ala Ala Ile Asn His Gly Leu Lys
Gly Leu385 390 395 400Gln Ile Ser Ala Ser Ala Trp Thr Ala Glu Ala
Leu Lys Leu Thr Met 405 410 415Pro Ala Ser Val Phe Ser Arg Ser Thr
Glu Cys His Asn Gln Asp Lys 420 425 430Val Ser Met Gly Thr Ile Ala
Ala Arg Asp Cys Leu Arg Val Leu Glu 435 440 445Leu Ala Glu Gln Val
Ala Ala Ala Leu Leu Ile Ala Val Arg Gln Gly 450 455 460Val Trp Leu
Arg Cys Arg Leu Asn Arg Ser Val Ala Pro Gly Ala Thr465 470 475
480Leu Lys Asn Met Met Asp Ala Leu Gly Ala Asp Ile Thr Val Ile Glu
485 490 495Glu Asp Arg Arg Leu Glu Pro Asp Leu Arg Leu Leu Leu Glu
Arg Ile 500 505 510Arg Gly Arg Ala Trp Asn Leu Tyr Glu 515
520471563DNAUnknownObtained from environmental sample 47atggatcccg
ctgacctgaa gacctcaagc ttacctcgga cgattgaatt cgaccaggga 60tggctgaaaa
tcgaagacat tgtcgacatc gccgaagggt cggctagggt ggcgttgtcc
120gatactgctg catttcgctc tggcattaag cgaggcgcgg ccttcctgga
ccgtctgctg 180cacgaagagg gcacgatcta tggcgttact accggctacg
gcgattcgtg cacggtgacg 240gtgccgcttg agctagtcgc cgagttgccg
cgccagctct atatctatca cggctgtggg 300ttgggcgact atctgagtcc
ggtacagacg cgcgccgtga tggcgacccg cctcacatcg 360ttatgcaaag
ggttttctgg cgtgagcttg gaactcctgc aacaaatcgt gaggttactc
420cagtgcgatt tattgccatt gattccgtcg gaaggctcgg tgggtgccag
cggcgatctc 480actccgctat cgtacctcgc tgcggttttg tgcggcgagg
gcgacgtttg gcgtaacggc 540gtccatgtaa gcgccgcgca ggcactggcc
gagggcggta ttacaccgct gtgtttgagg 600cccaaagaag gattggcgat
catgaatggg acggctgtca tgaccgcttt ggcctgcctt 660gctcatgtgc
gcgccgatta tctcacccga ctggttactc ggaccaccgc actggcgtcg
720tttgcgcttg acggcaatgc gcaccatttc gatgccacgt tgttcgcagt
gaaaccgcat 780ccaggcatgc agcgagtggc cggttggttg cgtcaagact
tgccctgcga tcacctggac 840cctaacagaa agcgtctgca ggaccgctat
tcgctccgct gcgcgcccca cgtgatcggt 900gttctcgccg acgcgctgcc
atggttgcgt gagtccatcg aaaatgaact caacagcgcc 960aacgacaatc
ccatcatcga tgcggaaggc caaggtgtcc tgtctggcgg tcacttttac
1020ggcgggcata tcgcgtttgc catggacagc atgaaaaatg ccgtcgcaaa
cctggcagat 1080ctgctcgatc ggcagatggc actgctggtc gatggccgct
acaacaatgg gctgccggtc 1140aacttgtccg gggctcaagg gcctcgcgcc
gcgatcaatc atgggctcaa ggggttacag 1200atcagcgctt cggcatggac
tgcggaagcg ctcaagttaa ccatgccggc ctcggtgttc 1260tcgcgctcga
ccgagtgcca taaccaggac aaagtcagtc tgggtacgat tgcagcacgc
1320gattgtttgc gggtgctcga attgacggag caagtcgccg ccgcgctgct
cattgcggta 1380cgacaaggag tctggctccg ttgcaggctg aatcgatccg
tcgctccggg aatggcgcta 1440aggaacatga tggatgccct gagcaccgat
atcaacgcga tagaggaaga tcgtcgactg 1500gagccggatt tgcgcttact
gctggagccc atccatagcc gtgtctggaa gctgtatgca 1560taa
156348520PRTUnknownObtained from environmental sample 48Met Asp Pro
Ala Asp Leu Lys Thr Ser Ser Leu Pro Arg Thr Ile Glu1 5 10 15Phe Asp
Gln Gly Trp Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Asp Thr Ala Ala Phe Arg Ser Gly 35 40
45Ile Lys Arg Gly Ala Ala Phe Leu Asp Arg Leu Leu His Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Ile Tyr 85 90 95His Gly Cys Gly Leu Gly Asp Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Asn Gly Val His Val Ser Ala Ala Gln Ala Leu Ala Glu Gly 180 185
190Gly Ile Thr Pro Leu Cys Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala His
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Thr Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
His Leu Asp Pro Asn Arg Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Leu Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Gln Gly Val Leu Ser
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Gly Arg Tyr Asn Asn Gly
Leu Pro Val Asn Leu Ser Gly 370 375 380Ala Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Val Ala Pro
Gly Met Ala Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asn Ala Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Pro Ile His 500 505 510Ser Arg Val Trp Lys
Leu Tyr Ala 515 520491563DNAUnknownObtained from environmental
sample 49atgcaccccg ctgaccagaa gaacgcaacc acagcccgta caattaaact
cgacaatggt 60cggctgagaa tcgaggacat tatggatatc gcggaggggt cggcccgtgt
cgtattgtcc 120gattcgcatg aattctgcgc cgccattaac cgaggggcgg
attttctcga ccgtttgctg 180cgtgaagagg gtacgatcta tggtgtcacc
accggctacg gcgattcgtg caccgtgacg 240gtcccggttg agctgatcgg
cgagctgccg cgccatctct acacctatca cggctgtggg 300ctgggcgact
atttgagtcc ggtacagaca cgcgcagtga tggcgactcg cctcacatcg
360ttgtgcaaag ggttctccgg cgtcagtctg gaactgctgc ggcaaatcgt
aaagttactc 420cagtgcgatc tattaccatt gattccatcc gaaggctcag
tgggtgccag cggtgatctc 480actccgttat cgtaccttgc tgccgttttg
tgcggcgagg gtgaagtttg gcgaaacggc 540gcccgtttaa gcgccgctca
ggcactgact gaggcagggg tgacaccgct gcggttgagg 600ccaaaagaag
gactggcgat catgaacggg acgtcggtaa tgaccggttt ggcttgcctt
660gcgtatgcac gtgccgatta tctcacccgg ctggtcactc gcatcaccgc
cctagcttcc 720tttgcgcttg acggcaacgc gcagcatttt gatcctaagt
tattctcggt gaagccgcat 780ccaggcctgc agcgcgtggc gggctggttg
cgtcaagact tgccgtgcga tcggcgtgac 840cgcgacggaa agcgtctaca
ggaccgttat tcggtgcgtt gcgcgcccca cgtgatcggt 900gttctagcag
acgccacgcc gtggctgcgc gactccatcg aaaatgaact caacagcgcc
960aatgacaatc ccatcattga tgcggaaggc gaaggcgtgc tgtatggtgg
ccacttttac 1020ggtgggcata tcgcgtttgc catggacagc atgaaaaatg
ccgtcgcaaa cctggcggat 1080ctgcttgacc ggcagatggc cctgctggtc
gacagccgct acaataatgg tttggcgccc 1140aacttgtctg gagctcatgg
gccacgtgca gcgatcaatc atgggctcaa gggcctgcag 1200attagtgctt
cggcatgggc agcggaagcg ctcaagttaa cgatgccggc gtcggtgttc
1260tcgcgttcga cggagtgcca caatcaggat aaagtcagca tgggcaccat
tgcggcacgg 1320gattctctgc gggtgctcga gctaaccgag caggtcgctg
ccgcgatgct gatagccgtg 1380cgacaaggtg tctggctgcg ttgccggctg
aaccgatcca tggctccgga ggcgacgctg 1440cggaacatga tggacgcgct
aggcgctgat atcccggtaa tcgaggagga ccgcaagcta 1500gagccggatc
tcagactaat gctggagcgc attcgcggtc gggcctggaa gctatatgaa 1560taa
156350520PRTUnknownObtained from environmental sample 50Met His Pro
Ala Asp Gln Lys Asn Ala Thr Thr Ala Arg Thr Ile Lys1 5 10 15Leu Asp
Asn Gly Arg Leu Arg Ile Glu Asp Ile Met Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Val Leu Ser Asp Ser His Glu Phe Cys Ala Ala 35 40
45Ile Asn Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Val Glu Leu Ile Gly Glu Leu Pro Arg His Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Asp Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Arg Gln Ile
Val Lys Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Ala Arg Leu Ser Ala Ala Gln Ala Leu Thr Glu Ala 180 185
190Gly Val Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ser Val Met Thr Gly Leu Ala Cys Leu Ala Tyr
Ala Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala Gln His
Phe Asp Pro Lys Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Arg Arg Asp Arg Asp Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Thr Pro Trp Leu Arg Asp Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Gly Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Ala Pro Asn Leu Ser Gly 370 375 380Ala His Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Ala Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Leu Glu Leu
435 440
445Thr Glu Gln Val Ala Ala Ala Met Leu Ile Ala Val Arg Gln Gly Val
450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Met Ala Pro Glu Ala
Thr Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Gly Ala Asp Ile
Pro Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp Leu Arg
Leu Met Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys Leu Tyr
Glu 515 520511566DNAUnknownObtained from environmental sample
51atgcatcccg ttgacttgaa gaagacctcc acctcagtcc gtacgattcg attcgacgat
60agatggctgc aaatcgggga cattgtcgat atcgcggaag ggccggctag cgtagcactg
120tccgatgctt ctgaattccg ctccgccatt gcgcgagggg cggattttct
ggaccgtctg 180ctgcgcgaag agggcacgat ttacggcgta accaccggct
acggcgattc atgcaccgtg 240acggtcccgc ttgagttggt cgccgagtta
ccgcgtcatc tctacacgta tcacggctgc 300gggctggggg agtatttaag
tccggtacag acgcgcgccg tgatcgccac ccgcctcacg 360tcgttgtgca
aaggattctc cggcgtcagt ctggaactgc tgcgtcaact cgtgaggcta
420ctgcagtgtg atttgttgcc attgattcca tctgaaggtt cggtgggcgc
cagcggcgat 480ctcactccgc tatcctatct caccgccgtg ttgtgcggcg
agggtgaagt ttggcgcaac 540ggcgttcacg cgagcgccgc ggacgcactg
gctgaggccg gtatcacacc actgcagctg 600cggccaaaag aggggctggc
gatcatgaat gggacgtcgg tcatgaccgg gttggcgtgt 660cttgcctacg
tgcgcgccga ttacctcacg cggctggtta cccgcatcac tgctttggcg
720tcgtttgccc tggacggcaa tgcgcaccat ttcgatgcta cgttgttctc
agtgaagccg 780catccaggcc agcagcgggt ggccggtggg ctgcgtcaag
atttggcgtg cgatcagctg 840gaccccaacg gaaagcgcct ccaggatcgc
tactcgatcc gttgcgcgcc ccacgtgatc 900ggtgttctcg ccgatgcgct
gccatggttg cgcgagtcta tcgaaaatga actcaacagc 960gccaacgata
atccgatcat cgacgcggag ggcgaacggg tgctctatgg cggtcacttc
1020tacggggggc atattggatt tgccatggac agcatgaaaa atgccgtcgc
aaacctggcg 1080gatctgcttg accggcagat ggccctgctg gtcgacagcc
gctacaataa tgggttgccg 1140gccaatttgt ctggggttca agggccacgt
gcagcgatca atcatggcct caaggggttg 1200cagatcagtg tttcggcatg
gaccgcggag gcgctcaagt tgaccatgcc ggcgtcggtg 1260ttctcgcgtt
cgaccgagtg ccacaaccag gacaaagtca gcatgggtac gattgcggca
1320cgcgattgtt tgcgtgtgct cgaattgacg gagcaagtcg ccgcggcgtt
gctaatcacc 1380gtgcgacagg gggtctggct ccgttgcagg ctgaatcgat
cagccgctcc cgaggcgacg 1440ttgaagaaaa tgatgaacgc cttgggcgct
gatatcgccg agattgagga ggaccgcagg 1500ctagagccgg atttgcgatt
actactggag cgcatccgcg accgtgtctg gaaattgtat 1560gaataa
156652521PRTUnknownObtained from environmental sample 52Met His Pro
Val Asp Leu Lys Lys Thr Ser Thr Ser Val Arg Thr Ile1 5 10 15Arg Phe
Asp Asp Arg Trp Leu Gln Ile Gly Asp Ile Val Asp Ile Ala 20 25 30Glu
Gly Pro Ala Ser Val Ala Leu Ser Asp Ala Ser Glu Phe Arg Ser 35 40
45Ala Ile Ala Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu
50 55 60Gly Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val65 70 75 80Thr Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg His
Leu Tyr Thr 85 90 95Tyr His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro
Val Gln Thr Arg 100 105 110Ala Val Ile Ala Thr Arg Leu Thr Ser Leu
Cys Lys Gly Phe Ser Gly 115 120 125Val Ser Leu Glu Leu Leu Arg Gln
Leu Val Arg Leu Leu Gln Cys Asp 130 135 140Leu Leu Pro Leu Ile Pro
Ser Glu Gly Ser Val Gly Ala Ser Gly Asp145 150 155 160Leu Thr Pro
Leu Ser Tyr Leu Thr Ala Val Leu Cys Gly Glu Gly Glu 165 170 175Val
Trp Arg Asn Gly Val His Ala Ser Ala Ala Asp Ala Leu Ala Glu 180 185
190Ala Gly Ile Thr Pro Leu Gln Leu Arg Pro Lys Glu Gly Leu Ala Ile
195 200 205Met Asn Gly Thr Ser Val Met Thr Gly Leu Ala Cys Leu Ala
Tyr Val 210 215 220Arg Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile
Thr Ala Leu Ala225 230 235 240Ser Phe Ala Leu Asp Gly Asn Ala His
His Phe Asp Ala Thr Leu Phe 245 250 255Ser Val Lys Pro His Pro Gly
Gln Gln Arg Val Ala Gly Gly Leu Arg 260 265 270Gln Asp Leu Ala Cys
Asp Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln 275 280 285Asp Arg Tyr
Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala 290 295 300Asp
Ala Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser305 310
315 320Ala Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Arg Val Leu
Tyr 325 330 335Gly Gly His Phe Tyr Gly Gly His Ile Gly Phe Ala Met
Asp Ser Met 340 345 350Lys Asn Ala Val Ala Asn Leu Ala Asp Leu Leu
Asp Arg Gln Met Ala 355 360 365Leu Leu Val Asp Ser Arg Tyr Asn Asn
Gly Leu Pro Ala Asn Leu Ser 370 375 380Gly Val Gln Gly Pro Arg Ala
Ala Ile Asn His Gly Leu Lys Gly Leu385 390 395 400Gln Ile Ser Val
Ser Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met 405 410 415Pro Ala
Ser Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys 420 425
430Val Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu
435 440 445Leu Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Thr Val Arg
Gln Gly 450 455 460Val Trp Leu Arg Cys Arg Leu Asn Arg Ser Ala Ala
Pro Glu Ala Thr465 470 475 480Leu Lys Lys Met Met Asn Ala Leu Gly
Ala Asp Ile Ala Glu Ile Glu 485 490 495Glu Asp Arg Arg Leu Glu Pro
Asp Leu Arg Leu Leu Leu Glu Arg Ile 500 505 510Arg Asp Arg Val Trp
Lys Leu Tyr Glu 515 520531563DNAUnknownObtained from environmental
sample 53atggatcccg ctgatctgaa ggcctcaacc ttacctcgga cgattgaatt
cgaccaggga 60tggctgaaaa tcgaagacat tgtcgacatc gccgaagggt cggctagggt
ggcgttgtcc 120gatactgctg catctcgctc tggcattaag cgaggcgcag
ccttcctgga ccgtctgctg 180cacgaagagg gcacgatcta cggcgttact
accggctacg gcgattcgtg cacggtgacg 240gtgccgcttg agctggtcgc
cgagttgccg cgccagctct atatctatca cggttgtggg 300ttgggcgagt
atctgagtcc ggtacagacg cgcgccgtga tggcgacccg cctcacatcg
360ttgtgcaaag ggttttctgg cgtgagcttg gaactgctgc aacaaatcgt
gaggttactc 420cagtgcgatt tattgccatt gattccgtcg gaaggctcgg
tgggtgccag cggcgatctc 480actccgctat cgtacctcgc tgcggttttg
tgcggcgagg gcgacgtttg gcgtaacggc 540gtccatgtaa gcgccgcgca
ggcactggcc gagggcggta tcacaccgct gcgtttgagg 600cccaaagaag
ggttggcgat catgaatggg acggctgtca tgaccgcttt ggcctgcctt
660gcttatgtgc gcgccgatta tctcacccga ctggttactc ggaccaccgc
actggcgtcg 720tttgcgcttg acggtaatgc ccaccatttc gacgccacgt
tgttcgcagt gaaaccgcat 780ccgggcatgc agcgagtggc cggttggttg
cgtcaagatt tgccgtgcga tcacctggac 840accaatagaa agcgtctgca
ggatcgctat tcgctccgct gcgcgcccca cgtgatcggt 900gttctagccg
acgcgctgcc atggttgcgc gagtccatcg aaaatgaact caacagcgct
960aacgacaatc ccatcatcga tgcggaaggc caaggtgtcc tgtctggcgg
tcatttttac 1020ggcgggcata tcgcatttgc catggacagc atgaaaaatg
ccgtcgctaa cctggcggat 1080ctgctcgatc ggcaaatggc actgctggtc
gatggccgct acaacaatgg gctgccggcc 1140aacttgtctg gggctcaagg
cccgcgcgcc gcgatcaatc atgggctcaa ggggttgcag 1200atcagcgctt
cggcatggac tgcggaagcg ctcaagttga ccatgccggc ctcggtgttc
1260tcgcgctcga ccgagtgcca taaccaggac aaagttagtc tgggtacgat
tgcagcacgc 1320gattgtttgc gggtgctcga attgacggag caagtcgccg
ccgcgctgct cattgccgta 1380cgacaaggag tctggctccg ttgcaggctg
aatcgatccg tcgctttggg aatggcgcta 1440aggaacatga tggatgccct
gagcaccgac atcaacgcga tagaggaaga tcgtcgactg 1500gagccggatt
tgcgattact cctggagcgc atccatagcc gtgtctggaa gctgtatgaa 1560taa
156354520PRTUnknownObtained from environmental sample 54Met Asp Pro
Ala Asp Leu Lys Ala Ser Thr Leu Pro Arg Thr Ile Glu1 5 10 15Phe Asp
Gln Gly Trp Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Asp Thr Ala Ala Ser Arg Ser Gly 35 40
45Ile Lys Arg Gly Ala Ala Phe Leu Asp Arg Leu Leu His Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Ile Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Asn Gly Val His Val Ser Ala Ala Gln Ala Leu Ala Glu Gly 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Thr Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
His Leu Asp Thr Asn Arg Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Leu Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Gln Gly Val Leu Ser
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Gly Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Ala Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Val Ala Leu
Gly Met Ala Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asn Ala Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile His 500 505 510Ser Arg Val Trp Lys
Leu Tyr Glu 515 520551563DNAUnknownObtained from environmental
sample 55atgcatgccg ctgagtcaaa gacatcagcc ttacctcgga cgattgtgtt
cgaccatgga 60tggctgaaaa tcaaagacat cgtcgacatt gcggaaggtt cggctagggt
cgcgttgtcc 120gaggctgctg catttcatgt tggcatcaag cgaggggcag
acttcctgga gcgtctgctc 180cgccaagagg gcacgatcta tggcgttact
acgggttacg gcgattcgtg cacggtgact 240gtccctcctg agatggtcgc
cgagttgccg cgccagctct acgtttatca cggctgcggg 300ctgggcgagt
atctgagtcc ggtgcagacg cgcgccgtga tggcgacccg cctcacgtcg
360ttgtgcaaag ggttttccgg cgtgagcctg gaactcctgc aacaaatcgt
aaagctactc 420cagcgcgatt tattgccgtt gattccgtcg gagggctcgg
taggtgccag tggcgacctg 480actccattat cgtatttcgc ggcggttttg
tgcggcgagg gcgacgtttg gcgcagcggc 540gtccatctca gggccactcg
ggcactggcc gaggccggca tcacgccgct gtgtctgaga 600cccaaagagg
gcttggcgat catgaatggg accgcggtca tgaccgcttt ggcctgcctt
660gcttatgtgc gcgccgatta tctcacccga ctggtcactc ggatcaccgc
gttggcgtct 720tttgcgcttg ccggcaatgc gcaccatttc gatgccgcgt
tgttcgccgt gaaaccgcat 780ccaggcatgc agcgagtggc cggttggttg
cgtcaagact tgttctcgga tcagttggac 840cccaacgaca agcgcctaca
ggatcgctat tcgatccgtt gcgcgcccca cgtgatcggt 900gttctagccg
atgcgctgcc gtggttgcgc gagtccattg aaaatgagct caacagcgcc
960aacgacaatc ccatcatcga tgcggtaggc caaggcgtgt tgtgtggcgg
tcacttttac 1020ggcgggcata tcgcatttgc catggacagc atgaaaaatg
ccgtcgccaa cctggcggac 1080ctgctcgacc ggcagatggc gctgttggtg
gatagccgct acagcaatgg gctgccagcc 1140aacttgtctg gggttcaagg
gccacgcgcc ccgatcaacc atgggcttaa ggggttgcag 1200atcggcgctt
cggcctggac tgccgaagcg ctcaagttga ccatgccggc atcggtcttc
1260tcgcgttcga ccgagtgcca taatcaggac aaagtcagcc tcggtactat
tgccgcacgc 1320gattgtttgc gtgtgctcga attgacggag caagttgccg
cggcgctgct cattgccgtg 1380cggcaaggag tctggctgcg ttgccgactc
aatcggtcct tggctccggc gaagacgcta 1440aaaaacatga tggacgcgct
agccgccgat atccgcgtga tcgaggagga tcgcaagctg 1500gaaccggatt
tgcgattact gttggagcgc atccgcgacc gcttctggaa gctctatgaa 1560taa
156356520PRTUnknownObtained from environmental sample 56Met His Ala
Ala Glu Ser Lys Thr Ser Ala Leu Pro Arg Thr Ile Val1 5 10 15Phe Asp
His Gly Trp Leu Lys Ile Lys Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Glu Ala Ala Ala Phe His Val Gly 35 40
45Ile Lys Arg Gly Ala Asp Phe Leu Glu Arg Leu Leu Arg Gln Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Pro Glu Met Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Lys Leu Leu Gln Arg Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Phe Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Ser Gly Val His Leu Arg Ala Thr Arg Ala Leu Ala Glu Ala 180 185
190Gly Ile Thr Pro Leu Cys Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Ala Gly Asn Ala His His
Phe Asp Ala Ala Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Phe Ser Asp
Gln Leu Asp Pro Asn Asp Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Val Gly Gln Gly Val Leu Cys
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Pro
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Gly Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Leu Ala Pro
Ala Lys Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ala Ala
Asp Ile Arg Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505
510Asp Arg Phe Trp Lys Leu Tyr Glu 515 520571566DNAUnknownObtained
from environmental sample 57atgcatcccg ttgacttgaa taagagctca
acctcagtcc gtatgattag gttcgacgat 60ggacggctga gaatcgagga tattgtcgat
atcgcggaag ggtcggctag tgtaacactg 120tccgatgctt gtgaattccg
ctccgccatt gcgcgagggg cggagtttct ggatcgtctg 180ctgcgcgaag
agggcacggt ttatggcgta accaccggct acggtgattc atgcaccgtg
240acggtgccgc ttgagttggt ggccgagttg ccacgccatc tctacactta
tcacggctgc 300gggctggggg agtatttaag tccggtacag acgcgggccg
tgatcgccac tcgcctcaca 360tcgttgtgca aaggactctc tggcgtcagt
ctggaactgc tgcatcaact cgtgaggtta 420ctgcagcacg atttattgcc
attgattcca tctgaaggtt cggtgggggc cagcggcgat 480ctcaccccgc
tatcgtacgt tgctgcggtt ttgtgcggcg aaggcgaagt ttggcgtaac
540ggtgtccatg taagcgccgc gcaggcactg catgaggccg ggctgacgcc
gctccggttg 600aggccaaaag aaggactggc catcatgaat gggacgtccg
tcatgaccgc tttggcgtgc 660cttgcttatg cgcgggccga gtacctcacg
cggctagtca ctcgcatcac cgcattggcg 720tcgtttgccc tggacggcaa
cgcgcaccat tttgatgcca agttattttc ggtgaagccg 780cacccaggcc
agcagcgggt ggcgagttgg ttgcggcaag acttagcgtg cgatcagctg
840gaccccaacg gaaagcgcct ccaggatcgc tactcgatcc gttgtgcgcc
ccacgtgatt 900ggtgttctcg ccgacgcact gccatggttg cgcgagttca
tcnaaagcga actcaacagc 960gccaacgaca atccgatcat cgacgcggag
ggcgaacgcg tgctcaatgg cggtcacttc 1020tatggggggc acattggatt
tgccatggac agcatgaaaa atgccatcgc aaacctggcg 1080gatcttcttg
accggcagat ggcactgctg gtcgacagtc gctacaataa tgggttgccg
1140gccaacttgt ctggggttca aggaccacgt gcagcgatca atcatggcct
caaggggttg 1200cagatcagtg cttcggcatg gactgcggag gcgctcaagt
tgaccatgcc ggcgtcggtg 1260ttctcgcgtt cgacggagtg ccacaaccag
gacaaagtca gcatgggtac cattgcggca 1320cgcgattgtt tgcgtgtgct
cgaattgacg gagcaagtcg ccgcggcgtt gctgatcacc 1380gtgcggcagg
gagtctggct ccgttgcagg ctgaatcgat ccgtcgttcc cgaaacaacg
1440ttgaagaaca tgatggaagc cttgggtgca gatatcgttg agatcgtgga
agatcgcagg 1500ctagagccgg atttgcggct agtgctggag cgtatccgtg
accgtgtctg gaaattgtat 1560gaataa 156658521PRTUnknownObtained from
environmental sample 58Met His Pro Val Asp Leu Asn Lys Ser Ser Thr
Ser Val Arg Met Ile1 5 10 15Arg Phe Asp Asp Gly Arg Leu Arg Ile Glu
Asp Ile Val Asp Ile Ala 20 25 30Glu Gly Ser Ala Ser Val Thr Leu Ser
Asp Ala Cys Glu Phe Arg Ser 35 40 45Ala Ile Ala Arg Gly Ala Glu Phe
Leu Asp Arg Leu Leu Arg Glu Glu 50 55 60Gly Thr Val Tyr Gly Val Thr
Thr Gly Tyr Gly Asp Ser Cys Thr Val65 70 75 80Thr Val Pro Leu Glu
Leu Val Ala Glu Leu Pro Arg His Leu Tyr Thr 85 90 95Tyr His Gly Cys
Gly Leu Gly Glu Tyr Leu Ser Pro Val Gln Thr Arg 100 105 110Ala Val
Ile Ala Thr Arg Leu Thr Ser Leu Cys Lys Gly Leu Ser Gly 115 120
125Val Ser Leu Glu Leu Leu His Gln Leu Val Arg Leu Leu Gln His Asp
130 135 140Leu Leu Pro Leu Ile Pro Ser Glu Gly Ser Val Gly Ala Ser
Gly Asp145 150 155 160Leu Thr Pro Leu Ser Tyr Val Ala Ala Val Leu
Cys Gly Glu Gly Glu 165 170 175Val Trp Arg Asn Gly Val His Val Ser
Ala Ala Gln Ala Leu His Glu 180 185 190Ala Gly Leu Thr Pro Leu Arg
Leu Arg Pro Lys Glu Gly Leu Ala Ile 195 200 205Met Asn Gly Thr Ser
Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Ala 210 215 220Arg Ala Glu
Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu Ala225 230 235
240Ser Phe Ala Leu Asp Gly Asn Ala His His Phe Asp Ala Lys Leu Phe
245 250 255Ser Val Lys Pro His Pro Gly Gln Gln Arg Val Ala Ser Trp
Leu Arg 260 265 270Gln Asp Leu Ala Cys Asp Gln Leu Asp Pro Asn Gly
Lys Arg Leu Gln 275 280 285Asp Arg Tyr Ser Ile Arg Cys Ala Pro His
Val Ile Gly Val Leu Ala 290 295 300Asp Ala Leu Pro Trp Leu Arg Glu
Phe Ile Xaa Ser Glu Leu Asn Ser305 310 315 320Ala Asn Asp Asn Pro
Ile Ile Asp Ala Glu Gly Glu Arg Val Leu Asn 325 330 335Gly Gly His
Phe Tyr Gly Gly His Ile Gly Phe Ala Met Asp Ser Met 340 345 350Lys
Asn Ala Ile Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln Met Ala 355 360
365Leu Leu Val Asp Ser Arg Tyr Asn Asn Gly Leu Pro Ala Asn Leu Ser
370 375 380Gly Val Gln Gly Pro Arg Ala Ala Ile Asn His Gly Leu Lys
Gly Leu385 390 395 400Gln Ile Ser Ala Ser Ala Trp Thr Ala Glu Ala
Leu Lys Leu Thr Met 405 410 415Pro Ala Ser Val Phe Ser Arg Ser Thr
Glu Cys His Asn Gln Asp Lys 420 425 430Val Ser Met Gly Thr Ile Ala
Ala Arg Asp Cys Leu Arg Val Leu Glu 435 440 445Leu Thr Glu Gln Val
Ala Ala Ala Leu Leu Ile Thr Val Arg Gln Gly 450 455 460Val Trp Leu
Arg Cys Arg Leu Asn Arg Ser Val Val Pro Glu Thr Thr465 470 475
480Leu Lys Asn Met Met Glu Ala Leu Gly Ala Asp Ile Val Glu Ile Val
485 490 495Glu Asp Arg Arg Leu Glu Pro Asp Leu Arg Leu Val Leu Glu
Arg Ile 500 505 510Arg Asp Arg Val Trp Lys Leu Tyr Glu 515
520591563DNAUnknownObtained from environmental sample 59atgcatgccg
ctgagtcaaa gacatcagcc ttacctcgga cgattgcgtt cgaccatgga 60tggctgaaaa
tcgaagacat cgccgacatt gcggaaggtt cggctagggt cgcgttgtcc
120gaggctgctg catttcatgt tggcatcaag cgaggggcag acttcctgga
gcgtctgctc 180cgccaagagg gcacgatcta tggcgttact acgggttacg
gcgattcgtg cacggtgact 240gtccctcctg agatggtcgc cgagttgccg
cgccagctct acgtttatca cggctgcggg 300ctgggcgagt atctgagtcc
ggtgcagacg cgcgccgtga tggcgacccg cctcacgtcg 360ttgtgcaaag
ggttttccgg cgtgagcctg gaactcctgc aacaaatcgt gaagctactc
420cagcgcgatt tattgccgtt gattccgtcg gagggctcgg tgggtgccag
tggcgacctg 480actccattat cgtacctcgc ggcggttttg tgcggcgagg
gcgacgtttg gcgcagcggc 540gtccatgtca gcgccactcg ggcactggcc
gaggccggca tcacaccgct gcgtctgaga 600cccaaagagg gcttggcgat
catgaatggg accgcggtca tgaccgcttt ggcctgcctt 660gcttatgtgc
gcgccgatta tctcacccga ctggtcactc ggatcaccgc gttggcgtcg
720tttgcgcttg acggcaatgc gcaccatttc gatgccgcgt tgttcgccgt
gaaaccgcat 780ccaggcatgc agcgagtggc cggttggttg cgtcaagact
tgttctcgga tcagttggac 840cccaacgaca agcgcctaca ggatcgctat
tcgatccgtt gcgcgcccca cgtgatcggt 900gttctagccg atgcgctgcc
ctggttgcgc gagtccattg aaaatgagct caacagcgcc 960aacgacaatc
ccatcatcga tgcggtaggc caaggcgtgt tgtgtggcgg tcacttttac
1020ggcgggcata tcgcatttgc catggacagc atgaaaaatg ccgtcgccaa
catggcggat 1080ctgctcgacc ggcagatggc gctgttggtg gatagccgct
acagcaatgg gctgccagcc 1140aacttgtctg gggttcaagg gccacgcgcc
ccgatcaacc atgggcttaa ggggttgcag 1200atcggcgctt cggcctggac
tgccgaagcg ctcaagttga ccatgccggc atcggtcttc 1260tcgcgttcga
ccgagtgcca taatcaggac aaagtcagcc tcggtactat tgccgcacgc
1320gattgtttgc gtgtgctcga attgacggag caagttgccg cggcgctgct
cattgccgtg 1380cggcaaggag tctggctgcg ttgccaactc aatcggtctt
tggctccggc gaagacgcta 1440aaaaacatga tggacgcgct agccgccgat
atccgcgtga tcgaggagga tcgcaagctg 1500gaaccggatt tgcgattact
gttggagcgc atccgcgacc gcttctggga gctgtatgaa 1560taa
156360520PRTUnknownObtained from environmental sample 60Met His Ala
Ala Glu Ser Lys Thr Ser Ala Leu Pro Arg Thr Ile Ala1 5 10 15Phe Asp
His Gly Trp Leu Lys Ile Glu Asp Ile Ala Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Glu Ala Ala Ala Phe His Val Gly 35 40
45Ile Lys Arg Gly Ala Asp Phe Leu Glu Arg Leu Leu Arg Gln Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Pro Glu Met Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Lys Leu Leu Gln Arg Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Ser Gly Val His Val Ser Ala Thr Arg Ala Leu Ala Glu Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Ala Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Phe Ser Asp
Gln Leu Asp Pro Asn Asp Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Val Gly Gln Gly Val Leu Cys
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Met Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Pro
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Gly Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Gln Leu Asn Arg Ser Leu Ala Pro
Ala Lys Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ala Ala
Asp Ile Arg Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Asp Arg Phe Trp Glu
Leu Tyr Glu 515 520611563DNAUnknownObtained from environmental
sample 61atgcatcccg ctgaccagaa gaacgcaacc acagcccgta caatcaaact
cgacaatggt 60cggctgagaa tcgaggacat tgtcgatata gcggaggggt cggcccgtgt
cgtattgtcc 120gattcgcatg aattctgcgc cgccattaac cgaggggcgg
attttctcga ccgtttgctg 180cgtgaagagg gtacgatcta tggtgtcacc
accggctacg gggattcctg cactgtgacg 240gtcccggttg agctggtcgg
cgagctgccg cgccatctct acacctatca cggctgtggg 300ctgggcgagt
atttgagtcc ggtacagaca cgcgcagtga tggcgactcg cctcacatcg
360ttgtgcaaag ggttctccgg cgtcagtctg gaactgctgc ggcaaatcgt
gcggttactc 420cagtacgatt tattgccatt gattccatcc gaaggctcag
tgggtgccag cggcgatctc 480actccgttgt cgtaccttgc tgccgttttg
tgcggcgagg gtgaagtttg gcgcagcggc 540gtccgtttaa gcgccgctca
ggcactgact gaggcaggga tgacagcact gcggttgagg 600ccaaaagaag
gactggcgat catgaatggg acgtcggtaa tgaccggttt ggcttgcctt
660gcctatgcac gtgccgatta tctcactcgc ctggtcactc gcatcaccgc
cctagcgtcc 720tttgcgcttg acggcaacgc acaccatttt gatgataagt
tattctcagt gaagccgcat 780ccaggcctgc agcgcgtggc gggctggttg
cgtcaagact tgcggtgcga tcagcgggac 840cgcaacggaa agcgtctgca
ggaccgttat tcggtgcgtt gcgcgcccca tgtaatcggt 900gttctagcag
acgcggcgcc gtggctgcgc gactccatcg aaaatgaact caacagcgcc
960aacgacaatc ccatcattga tgcggaaggc gaaggggtgc tgtatggggg
ccactgttac 1020ggtgggcata tcgcgttcgc catggacagc atgaaaaatg
ccgtcgcaaa tctggcggat 1080ctgcttgacc ggcagatggc cctgttggtc
gacagccgct acaacaatgg tctggcgccc 1140aacttgtctg gagctcatgg
gccaagtgca accatcaatc atgggctcaa gggcttgcag 1200attagtgcgt
cggcatgggc ggcggaagcg ctcaagttaa cgatgccggc gtcggtgttc
1260tcgcgttcga ccgagtgcca caatcaggac aaagtcagca tgggcaccat
cgcggcacgg 1320gattctctgc gggtgatcga gctaaccgaa caggtcgctg
ccgcgatgct gatagccgtg 1380cgacaaggtg tctggctgcg ttgccggcta
aaccgatcca tggctccgga ggcgacgctg 1440cggaacatga tggacgcgct
aggcgctgat atcccggtaa tcgaggagga ccgcaagcta 1500gagccggagc
tcagactaat gctggagcgc attcgcggtc ggtcctggaa gctatatgaa 1560taa
156362520PRTUnknownObtained from environmental sample 62Met His Pro
Ala Asp Gln Lys Asn Ala Thr Thr Ala Arg Thr Ile Lys1 5 10 15Leu Asp
Asn Gly Arg Leu Arg Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Val Leu Ser Asp Ser His Glu Phe Cys Ala Ala 35 40
45Ile Asn Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Thr65 70 75 80Val Pro Val Glu Leu Val Gly Glu Leu Pro Arg His Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Arg Gln Ile
Val Arg Leu Leu Gln Tyr Asp Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Ser Gly Val Arg Leu Ser Ala Ala Gln Ala Leu Thr Glu Ala 180 185
190Gly Met Thr Ala Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ser Val Met Thr Gly Leu Ala Cys Leu Ala Tyr
Ala Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Asp Lys Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Arg Cys Asp
Gln Arg Asp Arg Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Ala Pro Trp Leu Arg Asp Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Gly Val Leu Tyr
Gly 325 330 335Gly His Cys Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Ala Pro Asn Leu Ser Gly 370 375 380Ala His Gly Pro Ser Ala Thr
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Ala Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Ile Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Met Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Met Ala Pro
Glu Ala Thr Leu465 470 475 480Arg Asn Met Met Asp Ala Leu Gly Ala
Asp Ile Pro Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Glu
Leu Arg Leu Met Leu Glu Arg Ile Arg 500 505 510Gly Arg Ser Trp Lys
Leu Tyr Glu 515 520631563DNAUnknownObtained from environmental
sample 63atgcatgccg ctgagctgaa gacgccagcc tttccccgga cgattgcatt
cgaccatgga 60tggctgaaaa tcgaagacat tgtcgatatc gccgaaggtt cagctagggt
ggcgttgtcc 120gaggctgctg catttcactc tggcatcaag cgaggggcag
acttcctgga gcgtctgctt 180cgccaagagg gcacgatcta tggcgttact
acgggctacg gcgattcgtg catggtgacg 240gtcccgcctg agctggtcgc
cgagttgccg cgccagctct
atgtttatca cggctgtgga 300ctgggcgagt atttgagtcc ggtgcagacg
cgcgccgtga tggcgacccg cctgacatcg 360ttgtgcaaag ggttttctgg
cgtgagcctg gaattgctac aacaaatcgt gaggctactc 420cagtgcgatt
tgttgcctat gattccgtcg gaaggctcgg tgggtgccag cggcgatctc
480actccgttat cgtacctcgc ggcggttttg tgcggcgagg gcgacgtttg
gcgcaacggc 540gtccatgtca gcgccgcgca ggcgctggcc gaggccggta
tcgcaccgct gtgtttgagg 600cccaaagagg gattggcaat catgaatgga
acagcggtta tgaccgcttt ggcctgcctt 660gcttatgcgc gcgccgatta
tctcatccga ctggtcacgc ggatcaccgc gttggcgtcc 720tttgcgcttg
acggcaatgc gcgccatttc gatgccacgt tgttcgcagt aaaaccccat
780ccgggcatgc agcgagtggc gggttggttg cgtcaagact tgccgtccgg
tcagctggac 840cccaacagaa agcgtttaca ggatcgctat tcgatccgtt
gcgcgcccca cgtgatcggt 900gttctagccg acgcgctgcc atggttgcgc
gagtccatcg aaattgagct caacagcgcc 960aatgacaatc ctattatcga
tgcggcaagc caaggggtgt tgtctggcgg gcacttttat 1020ggcgggcata
tcgcgtttgc catggacagc atgaaaaatg ccgtcgccaa cctggcggat
1080ctgctcgacc ggcagatggc actgctggtg gatagccgct acaacaatgg
gctgccggcc 1140aacttgtctg gggttcaagg gccacgcgcc acgatcaacc
acgggctcaa ggggttgcag 1200atcagtgctt cggcctggac tgcagaagcg
ctcaagctaa ccatgccggc gtcggtcttc 1260tcgcgttcga ccgagtgtca
taatcaggac aaagtcagcc tgggtactat tgccgcacgc 1320gattgtctgc
gtgtgctcga attgacggag caagtcgccg cggcgctgct cattgccgtg
1380cgacaaggag tctggctgcg ttgcagactc aatcggtcct tggctccggc
gaagacgctg 1440aaacacatga tggacgacct agccgccgac atccaggtgg
tcgaagagga ccgtaggctc 1500gaaccggatc tgcgcttact gctggagcgc
atccgcggcc gtttctggaa actgtatgaa 1560taa
156364520PRTUnknownObtained from environmental sample 64Met His Ala
Ala Glu Leu Lys Thr Pro Ala Phe Pro Arg Thr Ile Ala1 5 10 15Phe Asp
His Gly Trp Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Arg Val Ala Leu Ser Glu Ala Ala Ala Phe His Ser Gly 35 40
45Ile Lys Arg Gly Ala Asp Phe Leu Glu Arg Leu Leu Arg Gln Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Met Val
Thr65 70 75 80Val Pro Pro Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Met Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val 165 170 175Trp
Arg Asn Gly Val His Val Ser Ala Ala Gln Ala Leu Ala Glu Ala 180 185
190Gly Ile Ala Pro Leu Cys Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Ala Arg 210 215 220Ala Asp Tyr Leu Ile Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala Arg His
Phe Asp Ala Thr Leu Phe Ala 245 250 255Val Lys Pro His Pro Gly Met
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Ser Gly
Gln Leu Asp Pro Asn Arg Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Ile Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Ala Ser Gln Gly Val Leu Ser
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Thr
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Leu Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Leu Ala Pro
Ala Lys Thr Leu465 470 475 480Lys His Met Met Asp Asp Leu Ala Ala
Asp Ile Gln Val Val Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Phe Trp Lys
Leu Tyr Glu 515 520651563DNAUnknownObtained from environmental
sample 65atgcaccccg ttaacctaaa aacctcaacc tcagcccgta cgattatatt
cgacaatgga 60ccgctgaaaa ttgaggacat tgtggacatc gcggagggat cagctagcgt
gacattgtcc 120gatgcttctg aattccgctc tgccatagcg cgaggggcgg
atttcctgga tcggttgctg 180cgccaagaag gcatgatcta tggcgtcacc
acgggctacg gcgattcgtg caccgtgccg 240gtcccgcttg agctggtcac
cgagttgccg cgtcagctct atgtctatca cggttgtgga 300ctgggcgagt
atttgagtcc ggtacagacg cgcgccgtaa ttgcaacccg cctcacgtcc
360ttgtctaagg ggttctctgg cgtcagtctg gaactgctgc aacagatggt
gacgttactg 420cagtgcgata tattgccgct gattccgtgt gaagggtcgg
tgggtgccag cggggatctc 480actccgctat cgtacctcgc cgctgttttg
tgtggcgagg gtgaagtttg gcacaacggc 540gtcaaagtag gcgcggcaca
ggcactgtct gacgctggtg tcacgccgct gcgactcaga 600ccaaaagaag
gactagccgt catgaacggg acggcggtca tgaccgcttt ggcgtgcctt
660gcttacgtgc gcgccgatta tctcagtcgg ctagtcactc gcattaccgc
gctagcgtcg 720tttgcgcttg acggcaatgc gcaccatttc gatgccacgt
tattctcagt gaagccacac 780ccaggccagc agcgcgtggc cagctggctg
cgtcaagact tgctgtgcga tcagctgcac 840tccaacggaa agtgtctgca
ggatcgctat tccatccgtt gcgcgcctca cgtgatcggc 900gttctcgccg
atgcgctacc atggttgcgc gagtcgatcg aaaatgaact taacagcgcc
960aatgataatc ccatcgtcga tgcggaaggc aaaaccgtac tgcacggcgg
tcatttttac 1020ggcggccata tcgcgtttgc catggacagc atgaaaaatg
ccgtcgccaa cctggcggat 1080ctgattgacc ggcagatggc actcttggtg
gatagccgtt acaacaatgg tttgccggcc 1140aacttgtctg gggttcaagg
gccacgtgcg gcgatcaatc atgggctcaa ggcgttgcag 1200atcagcgctt
cggcatggac agcggaagcg cttaagttga cgatgccggc gtcggtgttt
1260tcgcgttcga cggagtgcca caaccaggac aaagtcagca tgggtaccat
tgcggcacgc 1320gactgtttgc gggtgctcga attggtggag caagttgcag
cggcggtgct gatcgccgtg 1380cgacagggag tctggctccg ttgcagactg
aatcgatcgg cggctccggg ggcgcctctg 1440aagaacatga tggacgccct
gagcaccgat atcaacgtga tcgaggaaga tcgtcgactt 1500gagccggatt
tgcgactact gctagagcgg atccgtgacc gcacctggaa actgtatgaa 1560taa
156366520PRTUnknownObtained from environmental sample 66Met His Pro
Val Asn Leu Lys Thr Ser Thr Ser Ala Arg Thr Ile Ile1 5 10 15Phe Asp
Asn Gly Pro Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Ser Val Thr Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala 35 40
45Ile Ala Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Gln Glu Gly
50 55 60Met Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Pro65 70 75 80Val Pro Leu Glu Leu Val Thr Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Ser
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Met
Val Thr Leu Leu Gln Cys Asp Ile 130 135 140Leu Pro Leu Ile Pro Cys
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
His Asn Gly Val Lys Val Gly Ala Ala Gln Ala Leu Ser Asp Ala 180 185
190Gly Val Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Val Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Ser Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Ser Trp Leu Arg Gln 260 265 270Asp Leu Leu Cys Asp
Gln Leu His Ser Asn Gly Lys Cys Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Val Asp Ala Glu Gly Lys Thr Val Leu His
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Ile Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Ala Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Val Glu Gln Val Ala Ala Ala Val Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Ala Ala Pro
Gly Ala Pro Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asn Val Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Asp Arg Thr Trp Lys
Leu Tyr Glu 515 520671566DNAUnknownObtained from environmental
sample 67atgcatcccg ttgacttgaa gaagaactca aactcagtcc gtacgattca
cttcgacgat 60ggacggctga aagtcgagga tattgtcgat atcgcggaag ggtcggctag
ggtagcactg 120tccgatgctt ctgaattccg ccccgcgatt gcgcgagggg
cggactttct ggaccgtttg 180ctgcgcgaag agggcacgat ttatggcgtc
accaccggct acggcgattc atgcaccgtg 240acagtcccgc tggatttggt
cgccgagttg ccgcgccatc tctacactta ccacggttgc 300gggctgggag
agtatttaag tcccgtacag acgcgggccg tgatcgccac ccgcctcacg
360tcgttgtgca aaggattctc tggcgtcagt ctcgaactgc tgcatcaact
tgtgaggttg 420ctgcagtgcg atttattgcc attgattcca tctgaaggtt
cggtgggcgc cagcggcgat 480ctcaccccac tatcttatct cgctgccgtg
ttgtgcggcg agggcgaagt ttggcgcgaa 540ggcgtccata cgagcgccgt
gcaggcactg gccgaggccg gtatcgcacc gctacggttg 600aggccaaaag
aaggactggc gatcatgaat gggacgtcgg tcatgaccgc tttggcgtgc
660cttgcttatg cgcgcgccga ttacctcacc cggctagtca ctcgcatcac
cgcattggcg 720tcgtttgcgc tggacggcaa tgcgcaccat ttcgaggcta
cgttgttttt agtgaagccg 780cacccaggcc agcagcgggt ggccggttgg
ttgcgtcaag acttggcgtg cgatcagctg 840gaccccaacg gaaagcgtct
ccaggatcgc tactcgatcc gttgcgcgcc ccacgtgatc 900ggtgttctcg
ccgatgcgct gccatggttg cgcgagtcca tcgaaaatga actcaacagc
960gccaacgata atccgatcat cgacgcggag ggcgaacggg tgctctatgg
cggtcacttc 1020tacggggggc atattggatt tgccatggac agcatgaaaa
atgccgtcgc aaacctggcg 1080gatttgcttg accggcagat ggccctgctg
gtcgacagcc ggtacaataa tgggttgcca 1140gccaatttgt ctggggttca
agggccacgc gcagcgatca atcatggcct caaggggttg 1200cagatcagtg
tttcggcatg gactgcggag gcgctcaagt tgaccatgcc ggcgtcggtg
1260ttctcgcgtt cgaccgagtg ccacaaccag gacaaagtca gcatgggtac
gattgcggca 1320cgcgattgtt tgcgtgtgct cgaattgacg gagcaagtcg
ccgcggcgtt gctaatcacc 1380gtgcgacagg gggtttggct ccgttgcagg
ctgaatcgat cagccgctcc cgaggcgacg 1440ttgaagaaaa tgatggacgc
cttgggcgct gatatcgccg aggttgagga ggaccgcagg 1500ctagagccgg
atttgcgatt actactggag cgcatccgcg accgtgtctg ggaattgtat 1560gaataa
156668521PRTUnknownObtained from environmental sample 68Met His Pro
Val Asp Leu Lys Lys Asn Ser Asn Ser Val Arg Thr Ile1 5 10 15His Phe
Asp Asp Gly Arg Leu Lys Val Glu Asp Ile Val Asp Ile Ala 20 25 30Glu
Gly Ser Ala Arg Val Ala Leu Ser Asp Ala Ser Glu Phe Arg Pro 35 40
45Ala Ile Ala Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Glu
50 55 60Gly Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val65 70 75 80Thr Val Pro Leu Asp Leu Val Ala Glu Leu Pro Arg His
Leu Tyr Thr 85 90 95Tyr His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro
Val Gln Thr Arg 100 105 110Ala Val Ile Ala Thr Arg Leu Thr Ser Leu
Cys Lys Gly Phe Ser Gly 115 120 125Val Ser Leu Glu Leu Leu His Gln
Leu Val Arg Leu Leu Gln Cys Asp 130 135 140Leu Leu Pro Leu Ile Pro
Ser Glu Gly Ser Val Gly Ala Ser Gly Asp145 150 155 160Leu Thr Pro
Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu 165 170 175Val
Trp Arg Glu Gly Val His Thr Ser Ala Val Gln Ala Leu Ala Glu 180 185
190Ala Gly Ile Ala Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile
195 200 205Met Asn Gly Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala
Tyr Ala 210 215 220Arg Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile
Thr Ala Leu Ala225 230 235 240Ser Phe Ala Leu Asp Gly Asn Ala His
His Phe Glu Ala Thr Leu Phe 245 250 255Leu Val Lys Pro His Pro Gly
Gln Gln Arg Val Ala Gly Trp Leu Arg 260 265 270Gln Asp Leu Ala Cys
Asp Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln 275 280 285Asp Arg Tyr
Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala 290 295 300Asp
Ala Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser305 310
315 320Ala Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Arg Val Leu
Tyr 325 330 335Gly Gly His Phe Tyr Gly Gly His Ile Gly Phe Ala Met
Asp Ser Met 340 345 350Lys Asn Ala Val Ala Asn Leu Ala Asp Leu Leu
Asp Arg Gln Met Ala 355 360 365Leu Leu Val Asp Ser Arg Tyr Asn Asn
Gly Leu Pro Ala Asn Leu Ser 370 375 380Gly Val Gln Gly Pro Arg Ala
Ala Ile Asn His Gly Leu Lys Gly Leu385 390 395 400Gln Ile Ser Val
Ser Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met 405 410 415Pro Ala
Ser Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys 420 425
430Val Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu
435 440 445Leu Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Thr Val Arg
Gln Gly 450 455 460Val Trp Leu Arg Cys Arg Leu Asn Arg Ser Ala Ala
Pro Glu Ala Thr465 470 475 480Leu Lys Lys Met Met Asp Ala Leu Gly
Ala Asp Ile Ala Glu Val Glu 485 490 495Glu Asp Arg Arg Leu Glu Pro
Asp Leu Arg Leu Leu Leu Glu Arg Ile 500 505 510Arg Asp Arg Val Trp
Glu Leu Tyr Glu 515 520691563DNAUnknownObtained from environmental
sample 69atgcattcag ttgacctaag agcgtcggcc tctgttcgta caatcaaatt
cgataatgga 60cgcgtgaaaa tcgaggacat tgtcgatatt gcggaagggg cggcttgcgt
agtgttgtcc 120gatgctcccg aatttcgctc cgccattgtg cgaggcgcgg
atttcatcga ccgtgtgctt 180ggccaagagg gcacgatcta tggcgtcact
accggctacg gcgattcatg cacggtggcg 240atcccgcctg agttgatcgc
tgagttgccg cgccagcttt atacctatca cggctgcggg 300ctgggcgagc
acttgagtcc ggtacagacg agggcggtga ttgtgacccg gctcacgtcg
360ctgtgcaaag ggttctccgg cgtcagtctc gaactgctgc aacaaatcgt
gagattactc 420caaagcgacg tgttgccctt gattccagcg gaaggctcgg
tgggcgccag cggcgatctc 480actccgctat cgtacctcgc tgctgttttg
tgcggcgagg gtgaagtttg gtgcaacggt 540gtccaagtaa acgccgcgcg
ggccctgggg caggccggta taacaccgct gcggttgagg 600cccaaagaag
gattggcgat catgaacggg acggccgtca tgaccgctct tgcgtgcctt
660gcttatgtgc
gtgccgacta tcttacgcgg ctggtcactc gcatcactgc atcgacctcg
720tttgcgctcg acgggaatgc gcaccatttc gatgccacgt tgttctcagt
gaagccccat 780ccaggtctgc agcgcgtggc cgcttggttg cgccaagacc
tgccgtgcga tcggccagac 840ggcaacgcca agcgtctaca ggatcgttat
tctgtgcgct gcgcgcctca cgtgatcggt 900gttctagccg acgccctgcc
atggttgcgc gagtccatcg aaaacgagct caacagtgcc 960aacgacaatc
ctatcatcga tgcggaaggc gaaaaggtcc tgtacggtgg tcacttctac
1020ggcgggcata tcgcgtttgc catggacagc atgaaaaacg ccgttgccaa
cctggcggat 1080ctgctggacc ggcagatggc gttgctggtc gacagccgct
acaataatgg tttaccggcc 1140aatttgtctg gatctcaggg gccacgtgca
gcgatcaatc atgggcttaa gggcttacag 1200atcagcgctt cggcatggac
tgcggaagcg ctcaagttga ccatgccggc ttcagtgttc 1260tcgcgttcga
ccgagtgcca taatcaggac aaggtcagca tgggtacgat tgcggcacgt
1320gattctctac gcgtgctcga gttgacggag caagtcgccg cggcgttgct
gattgccgtg 1380cgacagggag tctggcttcg ttgcacaatg aatcgatccg
tcgctccgca ggcgacattg 1440aagaacatga tggacgcctt gggcgccgat
atcaccgtga tcgaagagga ccgcaagctg 1500gagccggatc tgcgattact
cctggagcgc atccgcggcc gtgcctggaa actgtatgaa 1560taa
156370520PRTUnknownObtained from environmental sample 70Met His Ser
Val Asp Leu Arg Ala Ser Ala Ser Val Arg Thr Ile Lys1 5 10 15Phe Asp
Asn Gly Arg Val Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ala Ala Cys Val Val Leu Ser Asp Ala Pro Glu Phe Arg Ser Ala 35 40
45Ile Val Arg Gly Ala Asp Phe Ile Asp Arg Val Leu Gly Gln Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Ala65 70 75 80Ile Pro Pro Glu Leu Ile Ala Glu Leu Pro Arg Gln Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu His Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Ile Val Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Ser Asp Val 130 135 140Leu Pro Leu Ile Pro Ala
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Cys Asn Gly Val Gln Val Asn Ala Ala Arg Ala Leu Gly Gln Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Ser Thr Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Ala Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Arg Pro Asp Gly Asn Ala Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Lys Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Ser Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Thr Met Asn Arg Ser Val Ala Pro
Gln Ala Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Gly Ala
Asp Ile Thr Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys
Leu Tyr Glu 515 520711557DNAUnknownObtained from environmental
sample 71atgcaccaag ttgattctga aaccaaactt cgcaccgttc gattcgatac
cgcgccggtg 60gccattgaag atgttgtcga tatcgcccat gggacagccg gtgcggcttt
gtctgacgca 120tcggagtttc gttcggcaat cacggctggc acggagtttc
tggaccggct gctggatggg 180gatcataaca tctatggtgt caccaccgga
tttggcgatt catgcacggt gaagatagag 240ccggacttaa ttgctgaact
gccgcagcac ctttacacgt atcatggttg cggcctcggt 300gactatctta
caccggagca aacgcgggcg gtgatcgccg tgcggctggc ttccttgtgc
360aaaggctatt ccggggtcag cgtcaatctt ttagaacaac tggtgaactt
gctccggcac 420gacctgcttc cggtgattcc ttccgaagga tcggtgggcg
ccagcggcga tctaactccg 480ctgtcgtatc tcgccgcaac gttatgcggc
gagggtgaag tgtggcgtga aggcacgcga 540atagctgcgc aacacgcgct
ggcggatgcc ggtctgacgc ctctgcgcct gcggccgaag 600gaaggactgg
ccatcatgaa cggcacgtcg gtgatgaccg ctctagcctg cctggcgtat
660ccgcgggcgg tatatctaac tcggctcgtc actcgtatta ccgctctcgc
ggtgtttgct 720ctcgacggta acgcctacca ttttgatgcc aaactatttt
ctatgaagcc gtatagaggc 780caacagcagg tggctgcttg gctgcggcgg
gatttgcggg agactcaacc gtccctggac 840agtacgcgcc tccaggatcg
ctactcaatt cgttgcgcgc cccacgtgat cggcgttctg 900gccgacgcgc
ttccctggct gcgggaatgg attgagagcg agctcaacag cgtaaacgac
960aatccgatca ttgatgcgga agacgagcgc gtcttgtacg gcggccactt
ctatggtggc 1020cacatcgctt ttgcaatgga cagcatgaaa aacgccgtgg
ccaacctggc cgatcttctc 1080gaccgtcaaa tggcgttgtt ggtggacagc
cgctacaaca acggcttgcc ggccaatttg 1140tccggcgcaa caggttcccg
catggcgatc aatcacgggc tcaagggatt gcagatcagc 1200gtgtctgcat
ggactgcgga agcgctcaag ctgactatgc cggcctcggt attttcacgc
1260tcaaccgaat gccataacca agacaaagtc agcatgggca gtatcgcggc
gcgagattgt 1320ctgcgcgtgc ttgaattaac cgagcaagtc gccgccggtt
tgctgatcgc cgtacgccag 1380ggagtctggc tgcgctgtag attgaatccc
gccgcagctc cggaggcgaa cgtaaaacca 1440atgctcgacg cgttggacgc
ggatttcgcg gtggtcgagg aagaccgcaa gcttgagcct 1500gaactgcgtg
tcttactgga aaggattcgg gggcgcgcct ggaagctcta tgaataa
155772518PRTUnknownObtained from environmental sample 72Met His Gln
Val Asp Ser Glu Thr Lys Leu Arg Thr Val Arg Phe Asp1 5 10 15Thr Ala
Pro Val Ala Ile Glu Asp Val Val Asp Ile Ala His Gly Thr 20 25 30Ala
Gly Ala Ala Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala Ile Thr 35 40
45Ala Gly Thr Glu Phe Leu Asp Arg Leu Leu Asp Gly Asp His Asn Ile
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Lys Ile
Glu65 70 75 80Pro Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr Thr
Tyr His Gly 85 90 95Cys Gly Leu Gly Asp Tyr Leu Thr Pro Glu Gln Thr
Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Val Asn
Leu Leu Arg His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Thr Leu Cys Gly Glu Gly Glu Val Trp Arg 165 170 175Glu
Gly Thr Arg Ile Ala Ala Gln His Ala Leu Ala Asp Ala Gly Leu 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Pro Arg
Ala Val 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu
Ala Val Phe Ala225 230 235 240Leu Asp Gly Asn Ala Tyr His Phe Asp
Ala Lys Leu Phe Ser Met Lys 245 250 255Pro Tyr Arg Gly Gln Gln Gln
Val Ala Ala Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Thr Gln Pro
Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Leu Arg Glu Trp Ile Glu Ser Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Ile Ile Asp Ala Glu Asp Glu Arg Val Leu Tyr Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Pro
Ala Asn Leu Ser Gly Ala Thr 370 375 380Gly Ser Arg Met Ala Ile Asn
His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Ala Ala Gly Leu Leu Ile Ala Val Arg Gln Gly Val
Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro Glu Ala
Asn Val Lys Pro465 470 475 480Met Leu Asp Ala Leu Asp Ala Asp Phe
Ala Val Val Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu Leu Arg
Val Leu Leu Glu Arg Ile Arg Gly Arg 500 505 510Ala Trp Lys Leu Tyr
Glu 515731557DNAUnknownObtained from environmental sample
73atggatcaag ttgattccaa aaccaaactt cgcacagttc ggttcgatgc agcgcgagtg
60accattgaag atattgtcga tatcgctcat agatcagccg gcgcggcttt atcggatgca
120tccgagtttc gttcgaccat tacgggcggc acggattttc tggaccggct
gttgcgtggg 180gagcatagca tttatggtgt cacgaccgga tttggtgatt
cgtgcacggt gaaaatagag 240ccggacttaa ttgccgaact cccacagcat
ctctacacgt accatggttg cggcctcggc 300gactatctta caccggagca
aacgcgggcg gtgatagccg tccggctggc ctccctgtgc 360aaaggttact
ccggggtcag cgtcaatctt ttagaacaac tggtgaactt gctctggcac
420gatctcctgc cggtgattcc ttccgaaggt tcggtgggcg ctagcggcga
tctaactccg 480ctgtcgtatc ttgctgcagc gttgtgcggc gagggtgaag
tgtggcacga aggcacgcga 540atagcagcgc gccaagcgct ggtggatgcc
ggtttaacgc cgttgcgttt gcggccgaag 600gaaggactgg ccatcatgaa
cgggacctcc gtgatgaccg ctctcgcctg cctggcgtat 660ccgcgggccg
aatatctaac tcggctcgtc acccgcatca ccgctctcgc tgtgtttgct
720ctcgacggca acgcctacca ttttgatgcc aaactatttt ctatgaagcc
gcatccaggc 780caacaacagg tggctgcttg gctgcggcgg gatttgcggg
agaatcaacc gtccctggat 840agcacgcgcc tccaggatcg ctattcaatt
cgctgcgcgc cccacgtcat cggcgttctg 900gccgacgcgc ttccgtggtt
gcgggaatgg atcgagaacg agcttaacag cgtaaacgac 960aatccgatca
ttgatgcgga agacgagcgt gttttgtacg gcggccattt ctatggcggc
1020catattgctt ttgcaatgga cagtatgaaa aacgccgtcg ccaacctagc
tgatcttctc 1080gaccgtcaaa tggctttgtt ggtggacagc cgctacaaca
atggcttggc agcgaatttg 1140tccggggcta cggattcccg catggcgatc
aatcatggac tcaagggatt gcagatcagt 1200gtttcggcat ggaccgcaga
agcgcttagg ttgactatgc ccgcgtcggt attttcgcgc 1260tcaacggaat
gccataatca ggacaaagtc agtatgggca gcatcgcggc acgcgattgt
1320ctgcgcgttc ttgaactaac cgagcaagct gtcgccgggt tgctaatcgc
cgtacgccaa 1380ggagtctggc tgcgctgtcg attgaatccg gccgccgctc
cacaggaaaa cgtacaacct 1440atgctcgacg ccttgggcgc ggatgtcgcg
gtgatcgagg aagaccgcaa gctcgagccg 1500gagctgcgtc ttttgctagc
tagaattcgg gagcgcgcct ggaagctcta tgaataa
155774518PRTUnknownObtained from environmental sample 74Met Asp Gln
Val Asp Ser Lys Thr Lys Leu Arg Thr Val Arg Phe Asp1 5 10 15Ala Ala
Arg Val Thr Ile Glu Asp Ile Val Asp Ile Ala His Arg Ser 20 25 30Ala
Gly Ala Ala Leu Ser Asp Ala Ser Glu Phe Arg Ser Thr Ile Thr 35 40
45Gly Gly Thr Asp Phe Leu Asp Arg Leu Leu Arg Gly Glu His Ser Ile
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Lys Ile
Glu65 70 75 80Pro Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr Thr
Tyr His Gly 85 90 95Cys Gly Leu Gly Asp Tyr Leu Thr Pro Glu Gln Thr
Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Val Asn
Leu Leu Trp His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Ala Leu Cys Gly Glu Gly Glu Val Trp His 165 170 175Glu
Gly Thr Arg Ile Ala Ala Arg Gln Ala Leu Val Asp Ala Gly Leu 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Pro Arg
Ala Glu 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu
Ala Val Phe Ala225 230 235 240Leu Asp Gly Asn Ala Tyr His Phe Asp
Ala Lys Leu Phe Ser Met Lys 245 250 255Pro His Pro Gly Gln Gln Gln
Val Ala Ala Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Asn Gln Pro
Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Leu Arg Glu Trp Ile Glu Asn Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Ile Ile Asp Ala Glu Asp Glu Arg Val Leu Tyr Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Ala
Ala Asn Leu Ser Gly Ala Thr 370 375 380Asp Ser Arg Met Ala Ile Asn
His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Arg Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Ala Val Ala Gly Leu Leu Ile Ala Val Arg Gln Gly Val
Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro Gln Glu
Asn Val Gln Pro465 470 475 480Met Leu Asp Ala Leu Gly Ala Asp Val
Ala Val Ile Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu Leu Arg
Leu Leu Leu Ala Arg Ile Arg Glu Arg 500 505 510Ala Trp Lys Leu Tyr
Glu 515751557DNAUnknownObtained from environmental sample
75atgcatcaag ttgattccaa aaccaaactt cgcacagttc ggttcgatac cgcgccagtg
60gcaattgaag acgttgtcga tatcgcccat gggacggctg gtgcgactct ctccgacgca
120ttcgagtttc gttccgcaat cactgccggc acggattttc tggaccggct
gttgcagggg 180gagcatagta tctatggtgt cacgaccgga ttcggtgatt
cgtgcacggt gaaaatagag 240gcggacttaa ttgccgaact gccacagcat
ctctacacgt accatagttg cggcctcggc 300gaatatctta caccggagca
aacgcgagcg gtgatagccg taaggctagc ctccctgtgc 360aaaggttact
ccggggtcag cgtcaatctt ttagaacaac tggtgaactt gctccggcac
420gatcttctgc cggtaattcc ttccgaagga tcggtgggcg ccagcggcga
tctaactccg 480ctatcgtatc tcgccgcagc gttgtgcgga gagggtgagg
tgtggcgcga aggaacgcga 540atagctgcac aacaagcgct ggcggatgcc
gctttaacgc cgttgcgttt gcggccgaag 600gaaggactgg ccatcatgaa
cggtacgtcg gtgatgaccg ctctggcctg cctggcgtat 660ccgcgggccg
aatatctaac tcggctcgtc acccgcatta ccgctctcgc tgtgtttgct
720ctcgacggca acgcctacca ttttgatgcc aaactatttt ctatgaagcc
gcatccaggc 780caacaacagg tggctgcttg gctgcggcgg gatttgcggg
agaatcaacc gtccctggac 840agcacgcgcc ttcaggatcg ctattcaatt
cgctgcgcgc cccacgtcat cggcgttctg 900gccgacgcgc ttccctggtt
gcgggaatgg atcgagaacg agcttaacag cgtaaatgac 960aatccgatca
ttaatgcgga ggacgagcgt gttttgtacg gcggccactt ctatggcggc
1020cacattgctt ttgcaatgga cagtatgaaa aacgccgtcg ctaacctagc
tgatcttctc 1080gaccgacaaa tggctttgtt ggtggacagc cgctacaaca
atggcttggc agccaatttg 1140tccggggcta ccggttcccg catggcgatc
aatcacgggc tcaagggatt gcagattagt 1200gtttcggcat
ggaccgcaga agcgcttaag ttgactatgc ccgcgtcggt gttttcgcgc
1260tcaacggaat gccataatca ggacaaagtc agtatgggca gcatcgcggc
acgcgattgt 1320ctgcgcgtcc ttgaattaac cgagcaagtt gtcgcagggt
tgctcatcgc cgtgcgccag 1380ggagtctggc tgcgctgtcg attgaatccg
gccgccgctc cagaggcgaa cgtacaacct 1440atgctcgacg ccttgggggc
ggacgtcgca gtggtcgagg aggaccgcaa gctcgagccg 1500gagctgcgtc
tcttgctgga aaggattcgg gagcgcgcct ggaagctcta tgaataa
155776518PRTUnknownObtained from environmental sample 76Met His Gln
Val Asp Ser Lys Thr Lys Leu Arg Thr Val Arg Phe Asp1 5 10 15Thr Ala
Pro Val Ala Ile Glu Asp Val Val Asp Ile Ala His Gly Thr 20 25 30Ala
Gly Ala Thr Leu Ser Asp Ala Phe Glu Phe Arg Ser Ala Ile Thr 35 40
45Ala Gly Thr Asp Phe Leu Asp Arg Leu Leu Gln Gly Glu His Ser Ile
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Lys Ile
Glu65 70 75 80Ala Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr Thr
Tyr His Ser 85 90 95Cys Gly Leu Gly Glu Tyr Leu Thr Pro Glu Gln Thr
Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Val Asn
Leu Leu Arg His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Ala Leu Cys Gly Glu Gly Glu Val Trp Arg 165 170 175Glu
Gly Thr Arg Ile Ala Ala Gln Gln Ala Leu Ala Asp Ala Ala Leu 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Pro Arg
Ala Glu 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu
Ala Val Phe Ala225 230 235 240Leu Asp Gly Asn Ala Tyr His Phe Asp
Ala Lys Leu Phe Ser Met Lys 245 250 255Pro His Pro Gly Gln Gln Gln
Val Ala Ala Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Asn Gln Pro
Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Leu Arg Glu Trp Ile Glu Asn Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Ile Ile Asn Ala Glu Asp Glu Arg Val Leu Tyr Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Ala
Ala Asn Leu Ser Gly Ala Thr 370 375 380Gly Ser Arg Met Ala Ile Asn
His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Val Ala Gly Leu Leu Ile Ala Val Arg Gln Gly Val
Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro Glu Ala
Asn Val Gln Pro465 470 475 480Met Leu Asp Ala Leu Gly Ala Asp Val
Ala Val Val Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu Leu Arg
Leu Leu Leu Glu Arg Ile Arg Glu Arg 500 505 510Ala Trp Lys Leu Tyr
Glu 515771557DNAUnknownObtained from environmental sample
77atgcaccaag ttgattctga aaccaaactt cgcaccgttc gattcgatac cgcgccggtg
60gccattgaag atgttgtcga tatcgcccat gggacagccg gtgcggcttt gtctgacgca
120tcggagtttc gttcggcaat cacggctggc acggagtttc tggactggct
gctggatggg 180gatcataaca tctatggtgt caccaccgga tttggcgatt
catgcacggt gaagatagag 240ccggacttaa ttgctgaact gccgcagcac
ctttacacgt atcatggttg cggcctcggt 300gactatctta catcggagca
aacgcgggcg gtgatcgccg tgcggctggc ttccttgtgc 360aaaggctatt
ccggggtcag cgtcaatctt ttagaacaac tggtgaactt gctccggcac
420gacctgcttc cggtgattcc ttccgaagga tcggtgggcg ccagcggcga
tctaactccg 480ctgtcgtatc tcgccgcagc gttatgcggc gagggtgaag
tgtggcgtga aggcacgcga 540atagctgcgc aacacgcgct ggcggatgcc
ggtctgacgc ctctgcgcct gcggccgaag 600gaaggactgg ccatcatgaa
cggcacgtcg gtgatgaccg ctctagcctg cctggcgtat 660cctcgggcgg
tatatctaac tcggctcgtc actcgtatta ccgctctcgc ggtgtttgct
720ctcgacggta gcgcctacca ttttgatgcc aaactatttt ctatgaagcc
gtatagaggc 780caacagcagg tggctgcttg gctgcggcgg gatttgcggg
agactcaacc gtccctggac 840agtacgcgcc tccaggatcg ctactcaatt
cgttgcgcgc cccacgtgat cggcgttctg 900gccgacgcgc ttccctggct
gcgggaatgg atcgagagcg agctcaacag cgtaaacgac 960aatccgatca
ttgatgcgga agacgagcgc gtcttgtacg gcggccactt ctatggtggc
1020cacgtcgctt ttgcaatgga cagcatgaaa aacgccgtgg ccaacctggc
cgatcttctc 1080gaccgtcaaa tggcgttgtt ggtggacagc cgctacaaca
acggcttgcc ggccaatttg 1140tccggcgcaa caggttcccg catggcgatc
aatcacgggc tcaagggatt gcagatcagc 1200gtgtctgcat ggactgcgga
agcgctcaag ctgactatgc cggcctcggt attttcacgc 1260tcaaccgaat
gccataacca agacaaagtc agcatgggca gtatcgcggc gcgggattgt
1320ctgcgcgtgc ttgaattaac cgagcaagtc gccgccggtt tgctgatcgc
cgtacgccag 1380ggagtctggc tgcgctgtag attgaatccc gccgcagctc
cggaggcgaa cgtaaaacca 1440atgctcgacg cgttggacgc ggatttcgcg
gtggtcgagg aagaccgcaa gcttgagcct 1500gaactgcgtg tcttactgga
aaggattcgg gggcgcgcct ggaagctcta tgaataa
155778518PRTUnknownObtained from environmental sample 78Met His Gln
Val Asp Ser Glu Thr Lys Leu Arg Thr Val Arg Phe Asp1 5 10 15Thr Ala
Pro Val Ala Ile Glu Asp Val Val Asp Ile Ala His Gly Thr 20 25 30Ala
Gly Ala Ala Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala Ile Thr 35 40
45Ala Gly Thr Glu Phe Leu Asp Trp Leu Leu Asp Gly Asp His Asn Ile
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Lys Ile
Glu65 70 75 80Pro Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr Thr
Tyr His Gly 85 90 95Cys Gly Leu Gly Asp Tyr Leu Thr Ser Glu Gln Thr
Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Val Asn
Leu Leu Arg His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Ala Leu Cys Gly Glu Gly Glu Val Trp Arg 165 170 175Glu
Gly Thr Arg Ile Ala Ala Gln His Ala Leu Ala Asp Ala Gly Leu 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Pro Arg
Ala Val 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu
Ala Val Phe Ala225 230 235 240Leu Asp Gly Ser Ala Tyr His Phe Asp
Ala Lys Leu Phe Ser Met Lys 245 250 255Pro Tyr Arg Gly Gln Gln Gln
Val Ala Ala Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Thr Gln Pro
Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Leu Arg Glu Trp Ile Glu Ser Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Ile Ile Asp Ala Glu Asp Glu Arg Val Leu Tyr Gly Gly
His 325 330 335Phe Tyr Gly Gly His Val Ala Phe Ala Met Asp Ser Met
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Pro
Ala Asn Leu Ser Gly Ala Thr 370 375 380Gly Ser Arg Met Ala Ile Asn
His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Ala Ala Gly Leu Leu Ile Ala Val Arg Gln Gly Val
Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro Glu Ala
Asn Val Lys Pro465 470 475 480Met Leu Asp Ala Leu Asp Ala Asp Phe
Ala Val Val Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu Leu Arg
Val Leu Leu Glu Arg Ile Arg Gly Arg 500 505 510Ala Trp Lys Leu Tyr
Glu 515791557DNAUnknownObtained from environmental sample
79atgcaccaag ttgattctga aaccaaactt cgcaccgttc gattcgatac cgcgccggtg
60gccattgaag atgttgtcga tatcgcccat gggacagccg gtgcggctct ctccgacgca
120ttcgagtttc gttcggcaat cacggcgggc acggagtttc tggaccggct
gctggatggg 180gatcataaca tctatggtgt caccaccgga tttggcgatt
cacgcacggt gaagatagag 240ccggacttaa ttgctgaact gccgcagcac
ctttacacgt accatggttg cggcctcggt 300gactatctta caccggagca
aacgcgggcg gtgatagccg tgcgactggc ttccttgtgc 360aaaggctatt
ccggggtcag cgtcaatctt ttagaacaac tggcgaactt gctccggcac
420gacctgcttc cagtgattcc ttccgaagga tcggtgggcg ccagcggcga
tctaactccg 480ctgtcgtatc tcgccgcagc gttatgcggc gagggtgaag
tgtggcgtga aggcacgcga 540atagctgcgc aacacgcgct ggcggatgcc
ggtctgacgc ctctgcgcct gcggccgaag 600gaaggactgg ccatcatgaa
cggcacgtcg gtgatgaccg ctctagcctg cctggcgtat 660ccgcgggcgg
tatatctaac tcggctcgtc actcgtatta ccgctctcgc ggtgtttgct
720ctcgacggta acgcctacca ttttgatgcc aaactatttt ccatgaagcc
gtatagaggc 780caacagcagg tagctgcttg gctgcggcgg gatttgcggg
agactcaacc gtccttggac 840agtacgcgcc tccaggatcg ctactcaatt
cgttgcgcgc cccatgtgat cggcgttctg 900gccgacgcgc ttccctggct
gcgggaatgg atcgagagcg agctcaacag cgtaaacgac 960aatccgatca
ttgatgcgga agacgagcgc gttttgtacg gcggccactt ctatggtggt
1020cacatcgctt ttgcaatgga cagcaagaaa aacgccgtgg ccaacctggc
cgatcttctc 1080gaccgtcaaa tggcgttgtt ggtggacagc cgctacaaca
acggcttgcc ggccaatttg 1140tccggggcta tgggttcccg gatggcgatc
aatcacgggc tcaagggatt gcagatcagc 1200gtgtctgcat ggactgcgga
agcgctcaag ctgactatgc cggcctcggt attttcacgc 1260tcaaccgaat
gccataacca agacaaagtc agcatgggca gtatcgcggc gcgggattgt
1320ctgcgcgtgc ttgaattaac cgagcaagtc gccgccggtt tgctgatcgc
cgtacgccag 1380ggagtctggc tgcgctgtag attgaatccc gccgcagctc
cggaggcgaa cgtaaaacca 1440atgctcgacg cgttggacgc ggatatcgcg
gtggtcgagg aagaccgcaa gcttgagcct 1500gagctgcgtg tcttactgga
aaggattcgg gggcgcgcct ggaagctcta tgaataa
155780518PRTUnknownObtained from environmental sample 80Met His Gln
Val Asp Ser Glu Thr Lys Leu Arg Thr Val Arg Phe Asp1 5 10 15Thr Ala
Pro Val Ala Ile Glu Asp Val Val Asp Ile Ala His Gly Thr 20 25 30Ala
Gly Ala Ala Leu Ser Asp Ala Phe Glu Phe Arg Ser Ala Ile Thr 35 40
45Ala Gly Thr Glu Phe Leu Asp Arg Leu Leu Asp Gly Asp His Asn Ile
50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Arg Thr Val Lys Ile
Glu65 70 75 80Pro Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr Thr
Tyr His Gly 85 90 95Cys Gly Leu Gly Asp Tyr Leu Thr Pro Glu Gln Thr
Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys Gly
Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Ala Asn
Leu Leu Arg His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu Gly
Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser Tyr
Leu Ala Ala Ala Leu Cys Gly Glu Gly Glu Val Trp Arg 165 170 175Glu
Gly Thr Arg Ile Ala Ala Gln His Ala Leu Ala Asp Ala Gly Leu 180 185
190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met Asn Gly
195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr Pro Arg
Ala Val 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr Ala Leu
Ala Val Phe Ala225 230 235 240Leu Asp Gly Asn Ala Tyr His Phe Asp
Ala Lys Leu Phe Ser Met Lys 245 250 255Pro Tyr Arg Gly Gln Gln Gln
Val Ala Ala Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Thr Gln Pro
Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser Ile Arg
Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295 300Pro
Trp Leu Arg Glu Trp Ile Glu Ser Glu Leu Asn Ser Val Asn Asp305 310
315 320Asn Pro Ile Ile Asp Ala Glu Asp Glu Arg Val Leu Tyr Gly Gly
His 325 330 335Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp Ser Lys
Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln
Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn Gly Leu Pro
Ala Asn Leu Ser Gly Ala Met 370 375 380Gly Ser Arg Met Ala Ile Asn
His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val Ser Ala Trp
Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410 415Val Phe
Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met 420 425
430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu Thr Glu
435 440 445Gln Val Ala Ala Gly Leu Leu Ile Ala Val Arg Gln Gly Val
Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro Glu Ala
Asn Val Lys Pro465 470 475 480Met Leu Asp Ala Leu Asp Ala Asp Ile
Ala Val Val Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu Leu Arg
Val Leu Leu Glu Arg Ile Arg Gly Arg 500 505 510Ala Trp Lys Leu Tyr
Glu 515811557DNAUnknownObtained from environmental sample
81atgccgcaag ttgattccaa aaccgaactt cgcacagttc ggttcgatac agcgcgcgtg
60accattgaag atattgtcga tgtcgcccgt ggatcggccg gcgcggcttt gtccgacgca
120ttcgagtttc gttcggcgat cgctgccggc acggattttc tggaccggct
gttgcatggg 180gagcatagca tctatggtgt cacgaccgga tttggcgatt
cgtgcacggt gaagatagag 240ccggacttaa ttgccgaact gccgcagcac
ctttacacgt accatggttg cggcctcggc 300gactatctta caccggagca
aacgcgggcg gtgatagccg tgcggctggc ctcgttgtgc 360aaaggctatt
ccggggtcag cgtcaatctt ttggaacaac tggtgaactt gctccggcac
420gatcttctgc cggtgattcc ttccgaagga tcggtgggcg ccagcggcga
tctaactccg 480ctttcatatc tcgccgcagc attgtgcgga gagggtgagg
tgtggcgtgg aggaacgcga 540atagccgcgc aacaagcgct ggtggatgcc
ggtttaacgc cgttgcgcct gcgaccgaag 600gaaggactcg ccatcatgaa
tggcacttcg gtgatgaccg ctctggcttg cctggcgtat 660ccgcgggcgg
aatatctaac tcggctcgtc actcgcatca ccgctcttgc tgtgtttgct
720ctcgacggca acgcctacca ttttgatgcc aaactatttt ccatgaagcc
gcatagaggc 780caacaacagg tggctgtttg gctgcggcgg gacttgcggg
agaatcaacc gtccttggac 840agcacgcgcc tccaggatcg ctattcaatt
cgctgtgcgc cccacgtcat cggcgttctg 900gccgatgcgc ttccctggct
gcgggaatgg atcgagaacg agcttaacag cgtaaacgac 960aatccgatca
ttgatgcgga agacgagcgt gttttatacg gcggccactt ctatggcggc
1020catgtcgctt ttgcaatgga cagcatgaaa aacgccgtcg ccaacctagc
cgatcttctc 1080gaccgtcaaa tggcgttgtt ggtggacagc cgctacaaca
acggcttgcc ggccaatttg 1140tcaggggcta cggattcccg catggcgatc
aatcacgggc tcaagggatt gcagatcagc 1200gtgtctgcat ggactgcgga
agcgctcaag ttgactatgc cggcctcggt gttttcacgc 1260tcaaccgaat
gccataatca ggacaaagtc agcatgggca gtattgcagc gcgggattgt
1320ctgcgggtgc ttgaattaac cgagcaagtc gccgcgggtt tgctgatcgc
cgtgcgccag 1380ggagtctggc tgcgctgtcg attgaatccc gccgccgctc
cgcaggcaaa cgtacaacct 1440atgctcgacg ccttgggcgc ggatgtcgcg
gtggtcgagg aagaccgcaa gctcgagccg 1500gagctgcgtc tcttgctgga
aaggattcgg gagcgcacct ggaagctcta tgaataa
155782518PRTUnknownObtained from environmental sample 82Met Pro Gln
Val Asp Ser Lys Thr Glu Leu Arg Thr Val Arg Phe Asp1 5 10 15Thr Ala
Arg Val Thr Ile Glu Asp Ile Val Asp Val Ala Arg Gly Ser 20 25 30Ala
Gly Ala Ala Leu Ser Asp Ala Phe Glu Phe Arg Ser Ala Ile Ala
35 40 45Ala Gly Thr Asp Phe Leu Asp Arg Leu Leu His Gly Glu His Ser
Ile 50 55 60Tyr Gly Val Thr Thr Gly Phe Gly Asp Ser Cys Thr Val Lys
Ile Glu65 70 75 80Pro Asp Leu Ile Ala Glu Leu Pro Gln His Leu Tyr
Thr Tyr His Gly 85 90 95Cys Gly Leu Gly Asp Tyr Leu Thr Pro Glu Gln
Thr Arg Ala Val Ile 100 105 110Ala Val Arg Leu Ala Ser Leu Cys Lys
Gly Tyr Ser Gly Val Ser Val 115 120 125Asn Leu Leu Glu Gln Leu Val
Asn Leu Leu Arg His Asp Leu Leu Pro 130 135 140Val Ile Pro Ser Glu
Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro145 150 155 160Leu Ser
Tyr Leu Ala Ala Ala Leu Cys Gly Glu Gly Glu Val Trp Arg 165 170
175Gly Gly Thr Arg Ile Ala Ala Gln Gln Ala Leu Val Asp Ala Gly Leu
180 185 190Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
Asn Gly 195 200 205Thr Ser Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Pro Arg Ala Glu 210 215 220Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Val Phe Ala225 230 235 240Leu Asp Gly Asn Ala Tyr His
Phe Asp Ala Lys Leu Phe Ser Met Lys 245 250 255Pro His Arg Gly Gln
Gln Gln Val Ala Val Trp Leu Arg Arg Asp Leu 260 265 270Arg Glu Asn
Gln Pro Ser Leu Asp Ser Thr Arg Leu Gln Asp Arg Tyr 275 280 285Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp Ala Leu 290 295
300Pro Trp Leu Arg Glu Trp Ile Glu Asn Glu Leu Asn Ser Val Asn
Asp305 310 315 320Asn Pro Ile Ile Asp Ala Glu Asp Glu Arg Val Leu
Tyr Gly Gly His 325 330 335Phe Tyr Gly Gly His Val Ala Phe Ala Met
Asp Ser Met Lys Asn Ala 340 345 350Val Ala Asn Leu Ala Asp Leu Leu
Asp Arg Gln Met Ala Leu Leu Val 355 360 365Asp Ser Arg Tyr Asn Asn
Gly Leu Pro Ala Asn Leu Ser Gly Ala Thr 370 375 380Asp Ser Arg Met
Ala Ile Asn His Gly Leu Lys Gly Leu Gln Ile Ser385 390 395 400Val
Ser Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro Ala Ser 405 410
415Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val Ser Met
420 425 430Gly Ser Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
Thr Glu 435 440 445Gln Val Ala Ala Gly Leu Leu Ile Ala Val Arg Gln
Gly Val Trp Leu 450 455 460Arg Cys Arg Leu Asn Pro Ala Ala Ala Pro
Gln Ala Asn Val Gln Pro465 470 475 480Met Leu Asp Ala Leu Gly Ala
Asp Val Ala Val Val Glu Glu Asp Arg 485 490 495Lys Leu Glu Pro Glu
Leu Arg Leu Leu Leu Glu Arg Ile Arg Glu Arg 500 505 510Thr Trp Lys
Leu Tyr Glu 515831563DNAUnknownObtained from environmental sample
83atgcaccccg ctgacctgaa aacctcaacc ttatcccgta cgattgaatt cgacactgga
60ccgctgaaaa tcgaggacat tgtggacatc gcggaagggt cggctagcgt gacattgtcc
120gatgcttctg aattccgctc tgcgattgcg cgcggggcgg actttctagc
ccgtctgttg 180cgcgaagagg gcgcaatcta tggcgtcacc accggctacg
gcgattcgtg cacggtgccg 240gtcccgcttg agctggtcgc cgagttgccg
cgtcagcttt atgtctatca cggctgcggg 300ctgggcgagt atctgagtcc
ggtacagacg cgcgccgtga tcgcgactcg cctcacgtcg 360ttgtccaaag
ggttctctgg cgtcagtctg gaactgctgc aacagatggt gagcttactg
420cagtgcaatt tattgccatt gattccgtct gaaggctcgg tgggtgccag
cggcgatctc 480actccgctat cgtacctcgc tgctgtcttg tgcggcgagg
gcgaagtttg gcgcaacggg 540gtccaggtag gcgccgcgcg ggccctggct
gaggctggta tcacaccgct gcgactccgg 600ccaaaagaag gactggccat
catgaatggg acggcggtca tgaccgcttt ggcgtgcctt 660gcttacatgc
gcgccgatta tctcacccgg ctagtcactc gcattaccgc attggcgtcg
720tttgcgcttg acggcaatgc gcaccatttc gatgctaggt tattctcagt
gaaaccgcat 780ccaggccagc agcgcgtggc tggttggctg cgtcaagact
tgccgtgcga tcagctggac 840cccaacggca agcggctgca ggatcgctat
tcgatccgct gcgcgcctca cgtgatcggc 900gttctcgccg acgcgttgcc
ctggttgcgc gagtccatcg aaaatgaact caacagcgcc 960aacgacaatc
ccatcgtcga tgcggaaggc gaaagcgtgc tgtatggcgg tcatttttac
1020ggcgggcata tcgcgtttgc tatggacagc atgaaaaatg ccgtcgcaaa
cctggcggat 1080ctgcttgacc ggcagatggc actgctggtg gatagccgct
acagcaatgg tttgccggcc 1140aacttgtctg gggttcaagg gccacgtgca
gcgatcaatc atgggctcaa ggggttgcag 1200atcagtgctt cggcatggac
agcggaggcg ctcaagttaa ccatgcccgc gtcagtgttc 1260tcgcgttcga
cggagtgcca taaccaggac aaagtcagca tgggtaccat tgcggcacgc
1320gattgtttgc gtgtgctcga attggtggag caagtcgccg cggcgctgct
gatcgctgtg 1380cgacaaggag tctggctacg ttgcaggttg aatggagccg
tggctcccgg ggcgacgttg 1440aagaacatga tggacgccct gagcgctgat
atcgacgtga tcgaggaaga tcgtcgattg 1500gagccggatt tgcgactcct
gctggagcgg atccgtggcc gcgtctggaa actgtatgaa 1560taa
156384520PRTUnknownObtained from environmental sample 84Met His Pro
Ala Asp Leu Lys Thr Ser Thr Leu Ser Arg Thr Ile Glu1 5 10 15Phe Asp
Thr Gly Pro Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Ser Val Thr Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala 35 40
45Ile Ala Arg Gly Ala Asp Phe Leu Ala Arg Leu Leu Arg Glu Glu Gly
50 55 60Ala Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Pro65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Ser
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Met
Val Ser Leu Leu Gln Cys Asn Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val Gln Val Gly Ala Ala Arg Ala Leu Ala Glu Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Met Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Arg Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Val Asp Ala Glu Gly Glu Ser Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Val Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Gly Ala Val Ala Pro
Gly Ala Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ser Ala
Asp Ile Asp Val Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Val Trp Lys
Leu Tyr Glu 515 520851545DNAUnknownObtained from environmental
sample 85atgacgacgc caacccatga gccggtaacc ttcggcgaac gccctttgcg
catcgaagac 60gtgctggccc tggccaaccg tcaggcaccc gtgcagttgc agagcgacgc
cgactaccgc 120gaacgcatcg ccaaaggcgc gcggtttctc gattcgctgc
tggacaagga aggcgtgatc 180tacggcgtga ccaccggtta cggcgactcc
tgcgtggtcg cggtgccgct gcatcacgtc 240gaagcgctgc cgcagcatct
ctacactttc cacggttgcg ggctgggcaa actgcttgac 300gcccaggcca
cccgtgcagt gctggcggcg cgtttgcagt cgctgtgcca cggcgtgtcc
360ggggtgcgca tcgagctgct ggaacgcctg catgccttcc tcgaacacga
catcctaccg 420ctgatccccg aagagggttc ggtgggcgcc agcggcgatc
tgacgccgct gtcctacgtc 480gccgcgaccc tttccggcga acgtgaagtg
atgttccgtg gcgagcgccg ccaggctgcc 540gatgtgcacc gagaactcgg
ctggaccccg ctggtgctgc gcccgaaaga agcgctggca 600ctgatgaacg
gcaccgccgt gatgaccggc ctcgcctgcc tggcctacgc ccgcgccgat
660tacctgctgc aactggccac gcgcatcacc gcgctgaacg tggtggcgct
gcaaggcaat 720ccggaacact tcgacgagcg tctgttcgcc gccaagccgc
acccggggca gatgcaggtc 780gccgcgtggc tgcgcaagga tctggcgatc
gacgcaccga ccgcgccgct gcatcgcctg 840caggatcgct actcgctgcg
ttgcgcaccg cacgtgctcg gcgtattggc cgacagcctg 900aactggctgc
gttcgttcat agagaccgaa ctcaacagcg ccaacgacaa cccgatcatc
960gacgccgaag ccgagcgcgt gctgcacggc gggcacttct acggcgggca
tatcgcgttc 1020gccatggaca gcctgaaaaa cctcgtggcc aacgtcgccg
acctgctcga ccggcaactc 1080gcgctgctgg tggacgagcg ttacaaccat
ggtttgccga gcaacctgtc cgccgccagc 1140gccgaccgtg cgatgctcaa
ccacggcttc aaggctgtgc agatcggcgc cagcgcctgg 1200actgccgaag
cgttgaaaaa caccatgccg gccagcgtct tctcgcgctc caccgaatgc
1260cacaaccagg acaaggtgag catgggcacc atcgccgccc gcgacgccat
tcgcgtgctg 1320gagctgaccg aacaggtcgc cgccgccaca ctgctcgcgg
ccaatcaggg cgtgtggctg 1380cgtggccgtg atgaagacgc acgaccactg
ccgccgtccc tggccgcgat gcacgaagcg 1440ctggccaaag acttcccgcc
ggtgatcgaa gaccgcgccc tggaaggcga actgcgcctg 1500tgcctgcaac
gcatcgccga acaacactgg aggttgcatg cgtag 154586514PRTUnknownObtained
from environmental sample 86Met Thr Thr Pro Thr His Glu Pro Val Thr
Phe Gly Glu Arg Pro Leu1 5 10 15Arg Ile Glu Asp Val Leu Ala Leu Ala
Asn Arg Gln Ala Pro Val Gln 20 25 30Leu Gln Ser Asp Ala Asp Tyr Arg
Glu Arg Ile Ala Lys Gly Ala Arg 35 40 45Phe Leu Asp Ser Leu Leu Asp
Lys Glu Gly Val Ile Tyr Gly Val Thr 50 55 60Thr Gly Tyr Gly Asp Ser
Cys Val Val Ala Val Pro Leu His His Val65 70 75 80Glu Ala Leu Pro
Gln His Leu Tyr Thr Phe His Gly Cys Gly Leu Gly 85 90 95Lys Leu Leu
Asp Ala Gln Ala Thr Arg Ala Val Leu Ala Ala Arg Leu 100 105 110Gln
Ser Leu Cys His Gly Val Ser Gly Val Arg Ile Glu Leu Leu Glu 115 120
125Arg Leu His Ala Phe Leu Glu His Asp Ile Leu Pro Leu Ile Pro Glu
130 135 140Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser
Tyr Val145 150 155 160Ala Ala Thr Leu Ser Gly Glu Arg Glu Val Met
Phe Arg Gly Glu Arg 165 170 175Arg Gln Ala Ala Asp Val His Arg Glu
Leu Gly Trp Thr Pro Leu Val 180 185 190Leu Arg Pro Lys Glu Ala Leu
Ala Leu Met Asn Gly Thr Ala Val Met 195 200 205Thr Gly Leu Ala Cys
Leu Ala Tyr Ala Arg Ala Asp Tyr Leu Leu Gln 210 215 220Leu Ala Thr
Arg Ile Thr Ala Leu Asn Val Val Ala Leu Gln Gly Asn225 230 235
240Pro Glu His Phe Asp Glu Arg Leu Phe Ala Ala Lys Pro His Pro Gly
245 250 255Gln Met Gln Val Ala Ala Trp Leu Arg Lys Asp Leu Ala Ile
Asp Ala 260 265 270Pro Thr Ala Pro Leu His Arg Leu Gln Asp Arg Tyr
Ser Leu Arg Cys 275 280 285Ala Pro His Val Leu Gly Val Leu Ala Asp
Ser Leu Asn Trp Leu Arg 290 295 300Ser Phe Ile Glu Thr Glu Leu Asn
Ser Ala Asn Asp Asn Pro Ile Ile305 310 315 320Asp Ala Glu Ala Glu
Arg Val Leu His Gly Gly His Phe Tyr Gly Gly 325 330 335His Ile Ala
Phe Ala Met Asp Ser Leu Lys Asn Leu Val Ala Asn Val 340 345 350Ala
Asp Leu Leu Asp Arg Gln Leu Ala Leu Leu Val Asp Glu Arg Tyr 355 360
365Asn His Gly Leu Pro Ser Asn Leu Ser Ala Ala Ser Ala Asp Arg Ala
370 375 380Met Leu Asn His Gly Phe Lys Ala Val Gln Ile Gly Ala Ser
Ala Trp385 390 395 400Thr Ala Glu Ala Leu Lys Asn Thr Met Pro Ala
Ser Val Phe Ser Arg 405 410 415Ser Thr Glu Cys His Asn Gln Asp Lys
Val Ser Met Gly Thr Ile Ala 420 425 430Ala Arg Asp Ala Ile Arg Val
Leu Glu Leu Thr Glu Gln Val Ala Ala 435 440 445Ala Thr Leu Leu Ala
Ala Asn Gln Gly Val Trp Leu Arg Gly Arg Asp 450 455 460Glu Asp Ala
Arg Pro Leu Pro Pro Ser Leu Ala Ala Met His Glu Ala465 470 475
480Leu Ala Lys Asp Phe Pro Pro Val Ile Glu Asp Arg Ala Leu Glu Gly
485 490 495Glu Leu Arg Leu Cys Leu Gln Arg Ile Ala Glu Gln His Trp
Arg Leu 500 505 510His Ala 871563DNAUnknownObtained from
environmental sample 87atgcatgccg ctgagttaaa gacatcaccc ttacctcgaa
cgattgcgtt cgaccatgga 60tggctgaaaa tcgaagacat cgtcgacatt gcggaaggtt
tggcaagggt cgcgttgtcc 120gaggctgctg catttcacgt tggcatcaag
cgaggggcag acttcctgga gcgtctgctc 180cgccaagagg gcacgatcta
tggcgttact acgggttacg gcgattcgtg cacggtgact 240gtccctcctg
agctagtcgc cgagttgccg cgccagctct acgtttatca cggctgcggg
300ctgggcgagt atctgagtcc ggtgcagacg cgcgccgtaa tggcgacccg
cctcacgtcg 360ttgtgcaaag ggttttccgg cgtgagcctg gaactcctgc
aacaaatcgt gaagctactc 420cagcgcgatt tattgccgtt gattccgtcg
gagggctcgg tgggtgccag tggcgacctg 480actccattat catacctcgc
ggcggtttta tgcggcgagg gcgacgtttg gcgcagcggt 540atccatgtca
gcgccgctcg gacactggcc gaggccggca ttacaccgct gcgtctgaga
600cccaaagagg gcttggcgat catgaatggg accgcggtca tgaccgcttt
ggcctgtctt 660gcttatgtgc gcgccgagta tctcacccga ctggtcaccc
ggatcaccgc gttggcgtcg 720tttgcgcttg acggcaatgc gcaacatttc
gatgccgcgt tgttcgccgt gaaaccacat 780ccaggcatgc agcgagtggc
cggttggttg cgtcaagact tgttctcgga tcagctggac 840cccaacgaca
agcgcctaca ggatcgctat tcgatccgtt gcgcgcccca cgtgatcggt
900gttctagccg acgcgctgtc ctggttccgc gagtccattg aaaacgagct
caacagcgcc 960gacgacaatc ccatcatcga tgcggtaggc caaggcgtgt
tgtgtggcgg tcacttttac 1020ggcgggcata tcgcatttgc catggacagc
atgaaaaatg cagtcgccaa cctggcggat 1080ctgctcgacc ggcagatggc
gctgttggtg gatagccgct acagcaatgg gctgccggcc 1140aacttgtctg
gggctcaagg gccacgcgcc ccgatcaacc atgggcttaa ggggttgcag
1200atcggtgctt cggcctggac tgccgaagcg ctcaagttga ccatgccggc
gtcggtcttc 1260tcgcgttcga ccgagtgcca taatcaggac aaagtcagcc
tcggtactct tgccgcacgc 1320gattgtttgc gtgtgctcga attgacggag
caagttgccg cggcgctgct cattgccgtg 1380cgacaaggag tctggctgcg
ttgccgactc aatcggtcct tggcttcggc gaggacgcta 1440aaaaacatga
tggacgcact agccgccgat atccgcgtga tcgaggagga tcgcaagctg
1500gaaccggatt tgcgattact gttggagcgc atccgcggcc gcttctggaa
gctgtatgaa 1560taa 156388520PRTUnknownObtained from environmental
sample 88Met His Ala Ala Glu Leu Lys Thr Ser Pro Leu Pro Arg Thr
Ile Ala1 5 10 15Phe Asp His Gly Trp Leu Lys Ile Glu Asp Ile Val Asp
Ile Ala Glu 20 25 30Gly Leu Ala Arg Val Ala Leu Ser Glu Ala Ala Ala
Phe His Val Gly 35 40 45Ile Lys Arg Gly Ala Asp Phe Leu Glu Arg Leu
Leu Arg Gln Glu Gly 50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly
Asp Ser Cys Thr Val Thr65 70 75 80Val Pro Pro Glu Leu Val Ala Glu
Leu Pro Arg Gln Leu Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu
Tyr Leu Ser Pro Val Gln Thr Arg Ala 100 105 110Val Met Ala Thr Arg
Leu Thr Ser Leu Cys Lys Gly Phe Ser Gly Val 115 120 125Ser Leu
Glu Leu Leu Gln Gln Ile Val Lys Leu Leu Gln Arg Asp Leu 130 135
140Leu Pro Leu Ile Pro Ser Glu Gly Ser Val Gly Ala Ser Gly Asp
Leu145 150 155 160Thr Pro Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly
Glu Gly Asp Val 165 170 175Trp Arg Ser Gly Ile His Val Ser Ala Ala
Arg Thr Leu Ala Glu Ala 180 185 190Gly Ile Thr Pro Leu Arg Leu Arg
Pro Lys Glu Gly Leu Ala Ile Met 195 200 205Asn Gly Thr Ala Val Met
Thr Ala Leu Ala Cys Leu Ala Tyr Val Arg 210 215 220Ala Glu Tyr Leu
Thr Arg Leu Val Thr Arg Ile Thr Ala Leu Ala Ser225 230 235 240Phe
Ala Leu Asp Gly Asn Ala Gln His Phe Asp Ala Ala Leu Phe Ala 245 250
255Val Lys Pro His Pro Gly Met Gln Arg Val Ala Gly Trp Leu Arg Gln
260 265 270Asp Leu Phe Ser Asp Gln Leu Asp Pro Asn Asp Lys Arg Leu
Gln Asp 275 280 285Arg Tyr Ser Ile Arg Cys Ala Pro His Val Ile Gly
Val Leu Ala Asp 290 295 300Ala Leu Ser Trp Phe Arg Glu Ser Ile Glu
Asn Glu Leu Asn Ser Ala305 310 315 320Asp Asp Asn Pro Ile Ile Asp
Ala Val Gly Gln Gly Val Leu Cys Gly 325 330 335Gly His Phe Tyr Gly
Gly His Ile Ala Phe Ala Met Asp Ser Met Lys 340 345 350Asn Ala Val
Ala Asn Leu Ala Asp Leu Leu Asp Arg Gln Met Ala Leu 355 360 365Leu
Val Asp Ser Arg Tyr Ser Asn Gly Leu Pro Ala Asn Leu Ser Gly 370 375
380Ala Gln Gly Pro Arg Ala Pro Ile Asn His Gly Leu Lys Gly Leu
Gln385 390 395 400Ile Gly Ala Ser Ala Trp Thr Ala Glu Ala Leu Lys
Leu Thr Met Pro 405 410 415Ala Ser Val Phe Ser Arg Ser Thr Glu Cys
His Asn Gln Asp Lys Val 420 425 430Ser Leu Gly Thr Leu Ala Ala Arg
Asp Cys Leu Arg Val Leu Glu Leu 435 440 445Thr Glu Gln Val Ala Ala
Ala Leu Leu Ile Ala Val Arg Gln Gly Val 450 455 460Trp Leu Arg Cys
Arg Leu Asn Arg Ser Leu Ala Ser Ala Arg Thr Leu465 470 475 480Lys
Asn Met Met Asp Ala Leu Ala Ala Asp Ile Arg Val Ile Glu Glu 485 490
495Asp Arg Lys Leu Glu Pro Asp Leu Arg Leu Leu Leu Glu Arg Ile Arg
500 505 510Gly Arg Phe Trp Lys Leu Tyr Glu 515
520891563DNAUnknownObtained from environmental sample 89atgcatccag
ttgacctaag agcgtcgacc cctgttcgta caattaaatt cgataatgga 60cgcgtgaaaa
tcgaggacat tgtcgatatt gcggaggggg cggcttgcgt agtgttgtcc
120aatgctcccg aattttgctc cgccattgtg cgaggcgcgg atttcatcga
ccgtgtgctt 180ggcgaagagg gcaccatcta tggcgtcact accggctacg
gcgattcatg cacggtggcg 240atcccgcttg agttgatcgc tgagttgccg
cgccagcttt atacctatca tggctgcggg 300ctgggcgagc atttgagtcc
gctacagacg agggcggtga ttgtgacccg gctcacgtcg 360ctgtgcaaag
ggttctccgg cgtcagtctc gaactgctgc aacaaatcgt gagattactc
420caaagcgacg tgttgccctt gattccagcg gaaggctcgg tgggcgccag
tggcgatctc 480actccgctat cgtacctcgc tgctgttttg tgcggcgagg
gtgaagtttg gtgcaacggt 540gtccaagtaa acgccgcgcg ggccctaggg
caggccggta taacaccgct gcggttgagg 600cccaaagaag gattggcgat
catgaacggg acggccgtca tgaccgctct tgcgtgcctt 660gcttatgtgc
gtgccgacta tcttacccgg ctcgttactc gcatcactgc attgacgccg
720tttgcgctcg acgggaatgc gcaccatttc gatgccacgt tgttctcagt
gaagccccat 780ccaggtctgc agcgcgtggc cgcttggttg cgccaagacc
tgccgtgcga tcggccagac 840ggcaacggca agcgtctaca ggatcgttat
tcggtgcgct gcgcgcctca cgtgatcggt 900gttctagccg acgccctgcc
atggttgcgc gagtccatcg aaaacgagct caacagtgcc 960aacgacaatc
ctatcatcga tgcggaaggc gaaaaggtcc tgtacggtgg tcacttctac
1020ggcgggcata tcgcgtttgc catggacagc atgaaaaacg ccgttgccaa
cctggcggat 1080ctgctggacc ggcagatggc gttgctggtc gacagccgct
acaacaatgg tttaccggcc 1140aatttgtctg gatctcaggg gccacgtgca
gcgatcaatc atgggcttaa gggcttacag 1200atcagcgctt cggcatggac
tgcggaagcg ctcaagttga ccatgccggc ttcagtgttc 1260tcgcgttcga
ccgagtgcca taatcaggac aaggtcagca tgggtacgat tgcggcacgt
1320gattctctac gcgtgctcga gttgacggag caagtcgccg cggcgttgct
gatcgccgtg 1380cgacagggag tctggcttcg ttgcagaatg aatcgatccg
tcgctccgca ggcgacattg 1440aagaacatga tggacgcctt gggcgccgat
atcaccgtga tcgaagagga ccgcaagctg 1500gagccggatc tgcgattact
cctggagcgc atccgcggcc gtgcctggaa actatatgaa 1560taa
156390520PRTUnknownObtained from environmental sample 90Met His Pro
Val Asp Leu Arg Ala Ser Thr Pro Val Arg Thr Ile Lys1 5 10 15Phe Asp
Asn Gly Arg Val Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ala Ala Cys Val Val Leu Ser Asn Ala Pro Glu Phe Cys Ser Ala 35 40
45Ile Val Arg Gly Ala Asp Phe Ile Asp Arg Val Leu Gly Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Ala65 70 75 80Ile Pro Leu Glu Leu Ile Ala Glu Leu Pro Arg Gln Leu
Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu His Leu Ser Pro Leu
Gln Thr Arg Ala 100 105 110Val Ile Val Thr Arg Leu Thr Ser Leu Cys
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Ile
Val Arg Leu Leu Gln Ser Asp Val 130 135 140Leu Pro Leu Ile Pro Ala
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Cys Asn Gly Val Gln Val Asn Ala Ala Arg Ala Leu Gly Gln Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Thr Pro225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Thr Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Leu
Gln Arg Val Ala Ala Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Asp
Arg Pro Asp Gly Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Val Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu Lys Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Ser Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Ser Leu Arg Val Leu Glu Leu
435 440 445Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Met Asn Arg Ser Val Ala Pro
Gln Ala Thr Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Gly Ala
Asp Ile Thr Val Ile Glu Glu 485 490 495Asp Arg Lys Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys
Leu Tyr Glu 515 520911566DNAUnknownObtained from environmental
sample 91atggatccag gtgacttgga aaagatctcg atctcgggtc ctacgattcg
attcgataac 60ggaccgctga agatcgagga cattgttgcc attgcggaag gggcggctag
cgtcgcatta 120tccgaagctc ctgaattccg ctgcgccatt tcgcgaggag
cggactttct cgaccgcctg 180ctacgcgaaa aggctacagt gtacggtgta
accactggct acggcgattc atgcactgta 240cccgtaccgg ttgagttgat
cgccgaatta ccgcgccatt tgtatatcta tcacggttgc 300ggattggggg
agtatttgag tccggtacaa acgcgcgccg tgatcgcgac gcgcctcgct
360tcgctgtgca aagggttttc gggcgtaagt ctggagctgc tccagcaact
cgtgggttta 420atacgatgca acatactgcc attggttcca tctgagggct
ccgtcggcgc cagtggcgat 480cttactccgc tatcttacat cgctgccgtt
ttgtgcggcg agggcgaagt ttggcgcgac 540ggcgtccatg tgcacgccgc
acaggcgttg gctgaggcgg gcatgacgcc attatggctg 600aggccaaagg
aaggattggc ggttatgaat ggaacggcag tcatgactgc tttggcctgc
660cttgcttacg tgcgcgccga ttatcttgct cgtctggtca ctcgcatcac
cgcattagcg 720tcgtttacgc ttgacggcaa tgcccagcac tttgatgcta
cattgttttc ggtgaagccg 780catccagggc agcagcgagt ggccgcttgg
ctgcgtcaag acttgccgtg cgatccgcag 840gaccccatcg gaaaacgtct
ccaggaccgc tattcgatcc gctgcgcgcc gcacgtgatt 900ggtgttctcg
ccgacgcact gccatggctg cgcgagttca tcgaaaacga actcaatagc
960gccaacgaca atcccattat cgacgccgag agggaagccg tgctgtacgg
gggtcacttc 1020catggcggac atattgcgtt cgcaatggac agcatgaaaa
atgccgtcgc aaatctggcg 1080gatctgcttg accgtcagat ggcactgata
gtcgacagcc gttacaataa tggtttgcct 1140gacaatctat ccggggttaa
aggcccacgt gcggccatca atcatggact gaaggcgttg 1200caaatcagtg
catcggcctg gactggggaa gccctcaagt tgaccatgcc ggcatcggtg
1260ttctcgcgtt caaccgagtg tcacaaccag gacaaagtca gcatgggtac
catcgcggcc 1320cgcgactgtt tgcgtgtgct cgaattgacg gagcaagttg
ctgccgctct actgattacc 1380gtgcgacagg gggtgtggct ccgttgcatg
gtgagtcgct ccgtcgttcc agaaacaaac 1440ttaaaaagga tgatggaggc
cgtaggcgct gacatccctg tgatcgggga ggaccgcagg 1500ttagagccgg
atttgcgcct actgttggaa cgcattcgtg gccgcgtttg gaaactatat 1560gaataa
156692521PRTUnknownObtained from environmental sample 92Met Asp Pro
Gly Asp Leu Glu Lys Ile Ser Ile Ser Gly Pro Thr Ile1 5 10 15Arg Phe
Asp Asn Gly Pro Leu Lys Ile Glu Asp Ile Val Ala Ile Ala 20 25 30Glu
Gly Ala Ala Ser Val Ala Leu Ser Glu Ala Pro Glu Phe Arg Cys 35 40
45Ala Ile Ser Arg Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg Glu Lys
50 55 60Ala Thr Val Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr
Val65 70 75 80Pro Val Pro Val Glu Leu Ile Ala Glu Leu Pro Arg His
Leu Tyr Ile 85 90 95Tyr His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro
Val Gln Thr Arg 100 105 110Ala Val Ile Ala Thr Arg Leu Ala Ser Leu
Cys Lys Gly Phe Ser Gly 115 120 125Val Ser Leu Glu Leu Leu Gln Gln
Leu Val Gly Leu Ile Arg Cys Asn 130 135 140Ile Leu Pro Leu Val Pro
Ser Glu Gly Ser Val Gly Ala Ser Gly Asp145 150 155 160Leu Thr Pro
Leu Ser Tyr Ile Ala Ala Val Leu Cys Gly Glu Gly Glu 165 170 175Val
Trp Arg Asp Gly Val His Val His Ala Ala Gln Ala Leu Ala Glu 180 185
190Ala Gly Met Thr Pro Leu Trp Leu Arg Pro Lys Glu Gly Leu Ala Val
195 200 205Met Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala
Tyr Val 210 215 220Arg Ala Asp Tyr Leu Ala Arg Leu Val Thr Arg Ile
Thr Ala Leu Ala225 230 235 240Ser Phe Thr Leu Asp Gly Asn Ala Gln
His Phe Asp Ala Thr Leu Phe 245 250 255Ser Val Lys Pro His Pro Gly
Gln Gln Arg Val Ala Ala Trp Leu Arg 260 265 270Gln Asp Leu Pro Cys
Asp Pro Gln Asp Pro Ile Gly Lys Arg Leu Gln 275 280 285Asp Arg Tyr
Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala 290 295 300Asp
Ala Leu Pro Trp Leu Arg Glu Phe Ile Glu Asn Glu Leu Asn Ser305 310
315 320Ala Asn Asp Asn Pro Ile Ile Asp Ala Glu Arg Glu Ala Val Leu
Tyr 325 330 335Gly Gly His Phe His Gly Gly His Ile Ala Phe Ala Met
Asp Ser Met 340 345 350Lys Asn Ala Val Ala Asn Leu Ala Asp Leu Leu
Asp Arg Gln Met Ala 355 360 365Leu Ile Val Asp Ser Arg Tyr Asn Asn
Gly Leu Pro Asp Asn Leu Ser 370 375 380Gly Val Lys Gly Pro Arg Ala
Ala Ile Asn His Gly Leu Lys Ala Leu385 390 395 400Gln Ile Ser Ala
Ser Ala Trp Thr Gly Glu Ala Leu Lys Leu Thr Met 405 410 415Pro Ala
Ser Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys 420 425
430Val Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu
435 440 445Leu Thr Glu Gln Val Ala Ala Ala Leu Leu Ile Thr Val Arg
Gln Gly 450 455 460Val Trp Leu Arg Cys Met Val Ser Arg Ser Val Val
Pro Glu Thr Asn465 470 475 480Leu Lys Arg Met Met Glu Ala Val Gly
Ala Asp Ile Pro Val Ile Gly 485 490 495Glu Asp Arg Arg Leu Glu Pro
Asp Leu Arg Leu Leu Leu Glu Arg Ile 500 505 510Arg Gly Arg Val Trp
Lys Leu Tyr Glu 515 520931563DNAUnknownObtained from environmental
sample 93atgcaccccg ctgacctgaa aacctcaacc ttatcccgta cgattgaatt
cgacactgga 60ccgctgaaaa tcgaggacat tgtggacatc gcggaagggt cggctagcgt
gacattgtcc 120gatgcttctg aattccgctc tgcgattgcg cgcggggcgg
actttctagc ccgtctgttg 180cgcgaagagg gcgcaaccta tggcgtcacc
accggctacg gcgattcgtg cacggtgccg 240gtcccgcttg agctggtcgc
cgagttgccg cgtcagcttt atgtctatca cggctgtggg 300ctgggcgagt
atctgagtcc ggtacagacg cgcgccgtga tcgcgactcg cctcacgtcg
360ttgtccaaag ggttctctgg cgtcagtctg gaactgctgc aacagatggt
gagcttactg 420cagtgcaatt tattgccatt gattccgtct gaaggctcgg
tgggcgccag cggcgatctc 480actccgctgt cgtacctcgc tgcggtcttg
tgcggcgagg gcgaagtttg gcgcaacggc 540gtccaggtag gtgccgcgcg
ggccctggct gaggctggta tcacaccgct gcgactccgg 600ccaaaagaag
gactggccat catgaatggg acggcggtca tgaccgcttt ggcgtgcctt
660gcttacgtgc gcgctgatta tctcaccagg ctagtcactc gcattaccgc
attggcgtcg 720tttgcactcg acggcaatgc gcaccatttc gatgctaggt
tattctcagt gaagccgcat 780ccaggccagc agcgggtggc tggttggctg
cgtcaagact tgccgtgcga acagctggac 840cccaacggca agcggctgca
ggatcgctat tccatccgtt gcgcgcccca cgtgatcggc 900gttctcgctg
acgcgctgcc ctggttgcgc gagtccatcg aaaatgaact caacagcgcc
960aacgacaatc ccatcgtcga tgcggaaggc gaaagcgtgc tgtatggcgg
tcatttttac 1020ggtgggcata tcgcgtttgc tatgaacagc atgaaaaatg
ccgtcgcaaa cctggcggat 1080ctgcttgacc ggcagatggc actgctggtg
gataaccgct acagcaatgg tttgccggcc 1140aacttgtctg gggttcaagg
gccacgtgca gcgatcaatc atgggctcaa ggggttgcag 1200atcagtgctt
cggcatggac agcggaggcg ctcaagttga ccatgcccgc gtcagtgttc
1260tcgcgttcga cggagtgcca taaccaggac aaagtcagca tgggtaccat
tgcggcacgc 1320gattgtttgc gtgtgctcga attggtggag caagtcgccg
cggcgctgct catcgctgtg 1380cgacaaggag tctggctccg ttgcaggttg
aatggagccg tggctccggg ggcggcgttg 1440aagaacatga tggacgccct
gagcaccgat atcgacgtga tcgaggaaga tcgtcgattg 1500gagccggatt
tgcgactact gctggagcgg atccgtggcc gcgtctggaa actgtatgaa 1560taa
156394520PRTUnknownObtained from environmental sample 94Met His Pro
Ala Asp Leu Lys Thr Ser Thr Leu Ser Arg Thr Ile Glu1 5 10 15Phe Asp
Thr Gly Pro Leu Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Ser Val Thr Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala 35 40
45Ile Ala Arg Gly Ala Asp Phe Leu Ala Arg Leu Leu Arg Glu Glu Gly
50 55 60Ala Thr Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val
Pro65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Ser
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Met
Val Ser Leu Leu Gln Cys Asn Leu 130 135 140Leu Pro Leu Ile Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val Gln Val Gly Ala Ala Arg Ala Leu Ala Glu Ala 180
185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly Asn Ala His His
Phe Asp Ala Arg Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Gly Trp Leu Arg Gln 260 265 270Asp Leu Pro Cys Glu
Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Val Asp Ala Glu Gly Glu Ser Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asn
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Leu Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Asn Arg Tyr Ser Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Ala
Ile Asn His Gly Leu Lys Gly Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Val Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Gly Ala Val Ala Pro
Gly Ala Ala Leu465 470 475 480Lys Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asp Val Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Val Trp Lys
Leu Tyr Glu 515 520951563DNAUnknownObtained from environmental
sample 95atgcacctcg ctgagctaaa aacctcaacc ccggcccgta cgattgaatt
cgacaatgga 60ccccggaaaa tcgaggacat tgtggacatt gcagaaggat cggcaagcgt
gacattgtct 120gatgcttccg aattccgctc cgccatcacg cgcggggcgg
actttctgga ccgtctggtg 180cgcgaagagg gcacaatcta tggtgtcacc
accggctacg gcgattcgtg cgccgtgccg 240gtgccgcttg agctggtcgc
cgagttgccg cgccagctct atgtctatca cggttgcggg 300ctgggcgagt
atttgagtcc ggtacagacg cgcgccgtaa tagcaacgcg cctcacttca
360ttgtccaagg gattctctgg cgtcagtctt gaattactcc aacagatggt
gaggttactg 420cagtgcgatt tattgccgtt gattccggct gaagggtcag
tgggcgccag tggcgatctc 480actccgctat cgtacctcgc cgcggtcttg
tgtggcgagg gtgaagtttg gcgcaacgga 540gtccaggtaa gcgcagcgca
ggcactgccc gaggctggga tcacaccgct acgactccgg 600ccaaaagaag
gactggcgat catgaatggc acagcggtca tgaccgcttt ggcgtgcctt
660gcttacgtgc gcgccgatta tcttacccgg ctagtgactc gcataaccgc
actatcgtca 720tttgcgctcg acagcaatgc ccaccatttc gatgctaaat
tgttctcggt gaagccgcat 780ccaggccagc agcgcgtggc caattggctg
cgtcaagatt tgtccagcaa tcagttggac 840cccaacggaa agcgtctgca
agatcgctat tcgatccgct gcgcgcctca cgtgatcggt 900gttctcgccg
atgcgctgcc ctggctacgc gagtcgattg aaaatgaact taatagcgcc
960aacgataatc ccatcgttga tgcagaagcc aaaaccgtat tgtatggggg
tcatttctac 1020ggagggcata tcgcgtttgc catggacagc atgaaaaatg
ccgtagcaaa tctggctgat 1080ctgattgacc gccaaatggc actcttggtg
gatagccgct acaacaatgg tttgccggcc 1140aacttgtctg gagttcaagg
gccacgtgcc acgatcaatc atggactcaa ggcgttgcag 1200atcagtgctt
cggcgtggac agcggaagcg cttaagttga cgatgccggc ttcggtgttc
1260tcgcgctcga cggagtgtca caaccaggac aaagtcagca tgggtacgat
tgcggcacgt 1320gattgtttac gtgtgctcga attggtggag caagtcgccg
cggcgcttct catcgctgtg 1380agacagggag tttggcttcg ttgtaggctg
aatcggtccg tgcttccggg agcaagctta 1440acgaatatga tggacgccct
gagcactgat atcaacgtca tagaagaaga tcgccgcctt 1500gagccggatt
tgcgactact actagagcgg atccgtggcc gcgcctggaa actctatgaa 1560taa
156396520PRTUnknownObtained from environmental sample 96Met His Leu
Ala Glu Leu Lys Thr Ser Thr Pro Ala Arg Thr Ile Glu1 5 10 15Phe Asp
Asn Gly Pro Arg Lys Ile Glu Asp Ile Val Asp Ile Ala Glu 20 25 30Gly
Ser Ala Ser Val Thr Leu Ser Asp Ala Ser Glu Phe Arg Ser Ala 35 40
45Ile Thr Arg Gly Ala Asp Phe Leu Asp Arg Leu Val Arg Glu Glu Gly
50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser Cys Ala Val
Pro65 70 75 80Val Pro Leu Glu Leu Val Ala Glu Leu Pro Arg Gln Leu
Tyr Val Tyr 85 90 95His Gly Cys Gly Leu Gly Glu Tyr Leu Ser Pro Val
Gln Thr Arg Ala 100 105 110Val Ile Ala Thr Arg Leu Thr Ser Leu Ser
Lys Gly Phe Ser Gly Val 115 120 125Ser Leu Glu Leu Leu Gln Gln Met
Val Arg Leu Leu Gln Cys Asp Leu 130 135 140Leu Pro Leu Ile Pro Ala
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155 160Thr Pro Leu
Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Glu Val 165 170 175Trp
Arg Asn Gly Val Gln Val Ser Ala Ala Gln Ala Leu Pro Glu Ala 180 185
190Gly Ile Thr Pro Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg 210 215 220Ala Asp Tyr Leu Thr Arg Leu Val Thr Arg Ile Thr
Ala Leu Ser Ser225 230 235 240Phe Ala Leu Asp Ser Asn Ala His His
Phe Asp Ala Lys Leu Phe Ser 245 250 255Val Lys Pro His Pro Gly Gln
Gln Arg Val Ala Asn Trp Leu Arg Gln 260 265 270Asp Leu Ser Ser Asn
Gln Leu Asp Pro Asn Gly Lys Arg Leu Gln Asp 275 280 285Arg Tyr Ser
Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp 290 295 300Ala
Leu Pro Trp Leu Arg Glu Ser Ile Glu Asn Glu Leu Asn Ser Ala305 310
315 320Asn Asp Asn Pro Ile Val Asp Ala Glu Ala Lys Thr Val Leu Tyr
Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala Phe Ala Met Asp
Ser Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala Asp Leu Ile Asp
Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ser Arg Tyr Asn Asn Gly
Leu Pro Ala Asn Leu Ser Gly 370 375 380Val Gln Gly Pro Arg Ala Thr
Ile Asn His Gly Leu Lys Ala Leu Gln385 390 395 400Ile Ser Ala Ser
Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro 405 410 415Ala Ser
Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp Lys Val 420 425
430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg Val Leu Glu Leu
435 440 445Val Glu Gln Val Ala Ala Ala Leu Leu Ile Ala Val Arg Gln
Gly Val 450 455 460Trp Leu Arg Cys Arg Leu Asn Arg Ser Val Leu Pro
Gly Ala Ser Leu465 470 475 480Thr Asn Met Met Asp Ala Leu Ser Thr
Asp Ile Asn Val Ile Glu Glu 485 490 495Asp Arg Arg Leu Glu Pro Asp
Leu Arg Leu Leu Leu Glu Arg Ile Arg 500 505 510Gly Arg Ala Trp Lys
Leu Tyr Glu 515 520971569DNAUnknownObtained from environmental
sample 97atgcaagccg ctgacgctaa gaccccatcc gctcaacatc tcgtgagctt
cgaccggggg 60cgtttgacga tagaagacat cgtcgccatt gccgcgcgca aggcgcgtgt
cgagctgtcg 120gccgatcccc agttccgcgc cgccattgcc aagggcgccg
acttcctcga ccgcctgctg 180cgcgaagacg gcacgatcta tggcgtgacc
accggctatg gcgattcctg taccgtgacc 240gtgccgccgg aactggtggc
cgaactgccg caccacctct acacctatca cggctgcggc 300ctcggtgagc
acttcacgcc ggaacagacg cgcgccatca tggccgcgcg cctggcttcg
360ctgtccaagg gtttttccgg cgtctcggtc gagttgctgg aacagatcgt
caagctgatg 420cagcacgact tgctgccggt gatgccgtcc gaaggctcgg
tcggcgccag cggcgacctg 480accccgctgt cttatctggc agccgtgctg
tgcggcgagc gcgaagtctg gcaagacggc 540aaacacgtcg aggccgccga
cgctttgcgc gcggccggca tcatcccttt gcgcctgcgc 600cccaaggaag
gcctggccat catgaacggc acggccgtca tgaccgcgct ggcttgcctg
660gcctatgacc gcgccgaata cctgacgcgc ctgtgcacac gcatcacggc
cttggcttcg 720ttcgcactcg acggcaacgc acaccatttc aatgaaacgc
tgttctcggt caagccgcac 780cccggcatgc agcnggtggc ggcctggctg
cgccatgact tgcccaccga cgtggtcgag 840cgcaacggca agcgtctgca
agaccgctat tcgatccgtt gcgcgccgca cgtgatcggc 900gtgctggccg
atgccttgcc cttcctgcga ctatcgattg aaaacgaact caacagcgcc
960aacgacaacc ccatcatcga tgccgaaggc gagcacgtgc tgcacggcgg
ccacttctac 1020ggcggccaca tcgcnttcgc catggatggc atgaagaatg
ccgtggccaa tctggccgat 1080ctgctcgacc ggcagatggc tttgctggtc
gatgcgcgtt acaaccacgg cttgccggcc 1140aacctgtccg gcgccgaagg
cccgcgcgct gcgatcaacc atggtttgaa agccttgcag 1200atcagcgcct
cggcctggac cgcggaagcc ttgaagctga cgatgccggc ctcggtgttt
1260tcgcgctcga ccgaatgcca caaccaggac aaggtcagca tgggcacgat
cgccgcgcgc 1320gattgcctgc gcgtcttgga actggtggaa caggtgacgg
ccgcgctcct gatcacggtg 1380cgccagggcg tgtggctgcg ccagcgcgtg
caacccgacc accagctgca tgtggcgctg 1440acggccatga tggcggagct
cggcgccgac gtgccggaag tgcgcgaaga ccggcgcctg 1500gaacccgatc
tgcgcctgat ggtcgaacgg atccgttccc aagcctggag cctgtatggc
1560caagcatga 156998522PRTUnknownObtained from environmental sample
98Met Gln Ala Ala Asp Ala Lys Thr Pro Ser Ala Gln His Leu Val Ser1
5 10 15Phe Asp Arg Gly Arg Leu Thr Ile Glu Asp Ile Val Ala Ile Ala
Ala 20 25 30Arg Lys Ala Arg Val Glu Leu Ser Ala Asp Pro Gln Phe Arg
Ala Ala 35 40 45Ile Ala Lys Gly Ala Asp Phe Leu Asp Arg Leu Leu Arg
Glu Asp Gly 50 55 60Thr Ile Tyr Gly Val Thr Thr Gly Tyr Gly Asp Ser
Cys Thr Val Thr65 70 75 80Val Pro Pro Glu Leu Val Ala Glu Leu Pro
His His Leu Tyr Thr Tyr 85 90 95His Gly Cys Gly Leu Gly Glu His Phe
Thr Pro Glu Gln Thr Arg Ala 100 105 110Ile Met Ala Ala Arg Leu Ala
Ser Leu Ser Lys Gly Phe Ser Gly Val 115 120 125Ser Val Glu Leu Leu
Glu Gln Ile Val Lys Leu Met Gln His Asp Leu 130 135 140Leu Pro Val
Met Pro Ser Glu Gly Ser Val Gly Ala Ser Gly Asp Leu145 150 155
160Thr Pro Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu Arg Glu Val
165 170 175Trp Gln Asp Gly Lys His Val Glu Ala Ala Asp Ala Leu Arg
Ala Ala 180 185 190Gly Ile Ile Pro Leu Arg Leu Arg Pro Lys Glu Gly
Leu Ala Ile Met 195 200 205Asn Gly Thr Ala Val Met Thr Ala Leu Ala
Cys Leu Ala Tyr Asp Arg 210 215 220Ala Glu Tyr Leu Thr Arg Leu Cys
Thr Arg Ile Thr Ala Leu Ala Ser225 230 235 240Phe Ala Leu Asp Gly
Asn Ala His His Phe Asn Glu Thr Leu Phe Ser 245 250 255Val Lys Pro
His Pro Gly Met Gln Xaa Val Ala Ala Trp Leu Arg His 260 265 270Asp
Leu Pro Thr Asp Val Val Glu Arg Asn Gly Lys Arg Leu Gln Asp 275 280
285Arg Tyr Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu Ala Asp
290 295 300Ala Leu Pro Phe Leu Arg Leu Ser Ile Glu Asn Glu Leu Asn
Ser Ala305 310 315 320Asn Asp Asn Pro Ile Ile Asp Ala Glu Gly Glu
His Val Leu His Gly 325 330 335Gly His Phe Tyr Gly Gly His Ile Ala
Phe Ala Met Asp Gly Met Lys 340 345 350Asn Ala Val Ala Asn Leu Ala
Asp Leu Leu Asp Arg Gln Met Ala Leu 355 360 365Leu Val Asp Ala Arg
Tyr Asn His Gly Leu Pro Ala Asn Leu Ser Gly 370 375 380Ala Glu Gly
Pro Arg Ala Ala Ile Asn His Gly Leu Lys Ala Leu Gln385 390 395
400Ile Ser Ala Ser Ala Trp Thr Ala Glu Ala Leu Lys Leu Thr Met Pro
405 410 415Ala Ser Val Phe Ser Arg Ser Thr Glu Cys His Asn Gln Asp
Lys Val 420 425 430Ser Met Gly Thr Ile Ala Ala Arg Asp Cys Leu Arg
Val Leu Glu Leu 435 440 445Val Glu Gln Val Thr Ala Ala Leu Leu Ile
Thr Val Arg Gln Gly Val 450 455 460Trp Leu Arg Gln Arg Val Gln Pro
Asp His Gln Leu His Val Ala Leu465 470 475 480Thr Ala Met Met Ala
Glu Leu Gly Ala Asp Val Pro Glu Val Arg Glu 485 490 495Asp Arg Arg
Leu Glu Pro Asp Leu Arg Leu Met Val Glu Arg Ile Arg 500 505 510Ser
Gln Ala Trp Ser Leu Tyr Gly Gln Ala 515 520991566DNAUnknownObtained
from environmental sample 99atgcatcccg ctgacctcac aacgaccgcg
cgcaccatcc cgttcagtca ggaacgtttg 60cgcatcgaag acatcgtcga tatcgccgcg
catgccgcac gcgtgacgct atcggacgat 120ccggcattcc gcgccaccat
cgcgcgcggc gccgatttcc tcgatcgcct cttgcaggaa 180gagggcgtga
tctatggcgt gacgactggc tatggcgact cctgtacggt cggcattccc
240gccgaactga tagcggagct gccgcatcgc ctgtacacct atcacggctg
cggcctcggc 300gactatttca cgccgcagca gacgcgcgcc atcatggcga
cgcgtctggc ttccctcagt 360aaggggtatt cgggcgtcag cgtcggcttg
ctggagcaga tcgtgcgctt gctcgacaag 420aacctgctgc cgctgattcc
ctcggaaggc tcagtgggtg ccagcggcga cctcacgccc 480ttgtcctatc
tcgccgccgt gttgtgcggc gaacgcgaag tgtggcgcga cggacggcag
540gtgagtgcgc aacaggcgct gaccgacgcc ggcatcgctc ctttacgcct
gcgtccgaag 600gaaggcctgg cactcatgaa cggcacggcc gtgatgaccg
cgctggcctg cctcgcttgg 660tcgcgtgcgg aatacctgac gaaactgtgc
gcacgcatca cggccatggc gtcctttgcg 720ctggacggca atgcgcatca
cttcgacgcc accttgttct cggtcaagcc gcacgctggc 780cagcaacgcg
ttgcggcgtg ggtgcgcagc gatctatcgc ttgcgcagac ggcacaacaa
840ggcaagcgct tgcaagaccg ttattcaatc cgttgcgcac cgcacgtgat
cggcgtgctg 900gctgacgcct tgccatggtt gcgcgagacg atcgaaaacg
agctgaacag cgccaacgac 960aatcccatca tcgatcccga aggcgagcgc
gtgctgcatg gcggtcactt ttacggcggt 1020catatcgcct tcgcgatgga
cagcatgaag aatgccgtcg ccaacctggc cgacctgatg 1080gatcggcaaa
tggccttgct ggtggatagt cgctacaaca acggcctgcc gtccaacttg
1140tccggcatgc agggaccatg cgctgccatc agtcacggct tgaaggcctt
gcagatcagc 1200gcctccgcct ggactgcgga agcgctcaag cagaccatgc
cggcctcggt gttttcgcgt 1260tcgaccgaat gtcacaacca ggacaaggtt
agcatgggta ccatcgccgc gcgcgattgc 1320ctgcgcgtgc tggaactgac
ggagcaggtt gccgccgcct tgctcatcac ggtgcagcag 1380ggcaccttgc
tgcgccagcg cgtgaatccg gaactcgtgc cagaagcgtc gctacgtgca
1440atgagcgaaa cgctgggcac cgatatcgcg atggtcgaag aagaccgccg
cctggaaccg 1500gatttgcgtc tgctgctgga gcggatccag tcatgcgcat
ggagcctgta tgaaaacgtc 1560gcctga 1566100521PRTUnknownObtained from
environmental sample 100Met His Pro Ala Asp Leu Thr Thr Thr Ala Arg
Thr Ile Pro Phe Ser1 5 10 15Gln Glu Arg Leu Arg Ile Glu Asp Ile Val
Asp Ile Ala Ala His Ala 20 25 30Ala Arg Val Thr Leu Ser Asp Asp Pro
Ala Phe Arg Ala Thr Ile Ala 35 40 45Arg Gly Ala Asp Phe Leu Asp Arg
Leu Leu Gln Glu Glu Gly Val Ile 50 55 60Tyr Gly Val Thr Thr Gly Tyr
Gly Asp Ser Cys Thr Val Gly Ile Pro65 70 75 80Ala Glu Leu Ile Ala
Glu Leu Pro His Arg Leu Tyr Thr Tyr His Gly 85 90 95Cys Gly Leu Gly
Asp Tyr Phe Thr Pro Gln Gln Thr Arg Ala Ile Met 100 105 110Ala Thr
Arg Leu Ala Ser Leu Ser Lys Gly Tyr Ser Gly Val Ser Val 115 120
125Gly Leu Leu Glu Gln Ile Val Arg Leu Leu Asp Lys Asn Leu Leu Pro
130 135 140Leu Ile Pro Ser Glu Gly Ser Val Gly Ala Ser Gly Asp Leu
Thr Pro145 150 155 160Leu Ser Tyr Leu Ala Ala Val Leu Cys Gly Glu
Arg Glu Val Trp Arg 165 170 175Asp Gly Arg Gln Val Ser Ala Gln Gln
Ala Leu Thr Asp Ala Gly Ile 180 185 190Ala Pro Leu Arg Leu Arg Pro
Lys Glu Gly Leu Ala Leu Met Asn Gly 195 200 205Thr Ala Val Met Thr
Ala Leu Ala Cys Leu Ala Trp Ser Arg Ala Glu 210 215 220Tyr Leu Thr
Lys Leu Cys Ala Arg Ile Thr Ala Met Ala Ser Phe Ala225 230 235
240Leu Asp Gly Asn Ala His His Phe Asp Ala Thr Leu Phe Ser Val Lys
245 250
255Pro His Ala Gly Gln Gln Arg Val Ala Ala Trp Val Arg Ser Asp Leu
260 265 270Ser Leu Ala Gln Thr Ala Gln Gln Gly Lys Arg Leu Gln Asp
Arg Tyr 275 280 285Ser Ile Arg Cys Ala Pro His Val Ile Gly Val Leu
Ala Asp Ala Leu 290 295 300Pro Trp Leu Arg Glu Thr Ile Glu Asn Glu
Leu Asn Ser Ala Asn Asp305 310 315 320Asn Pro Ile Ile Asp Pro Glu
Gly Glu Arg Val Leu His Gly Gly His 325 330 335Phe Tyr Gly Gly His
Ile Ala Phe Ala Met Asp Ser Met Lys Asn Ala 340 345 350Val Ala Asn
Leu Ala Asp Leu Met Asp Arg Gln Met Ala Leu Leu Val 355 360 365Asp
Ser Arg Tyr Asn Asn Gly Leu Pro Ser Asn Leu Ser Gly Met Gln 370 375
380Gly Pro Cys Ala Ala Ile Ser His Gly Leu Lys Ala Leu Gln Ile
Ser385 390 395 400Ala Ser Ala Trp Thr Ala Glu Ala Leu Lys Gln Thr
Met Pro Ala Ser 405 410 415Val Phe Ser Arg Ser Thr Glu Cys His Asn
Gln Asp Lys Val Ser Met 420 425 430Gly Thr Ile Ala Ala Arg Asp Cys
Leu Arg Val Leu Glu Leu Thr Glu 435 440 445Gln Val Ala Ala Ala Leu
Leu Ile Thr Val Gln Gln Gly Thr Leu Leu 450 455 460Arg Gln Arg Val
Asn Pro Glu Leu Val Pro Glu Ala Ser Leu Arg Ala465 470 475 480Met
Ser Glu Thr Leu Gly Thr Asp Ile Ala Met Val Glu Glu Asp Arg 485 490
495Arg Leu Glu Pro Asp Leu Arg Leu Leu Leu Glu Arg Ile Gln Ser Cys
500 505 510Ala Trp Ser Leu Tyr Glu Asn Val Ala 515
520101807DNAUnknownObtained from an environmental sample
101atgctttttg gcatttatcc acgttggaca cacctgatgc atcccgctga
cctgaagacc 60tcaaccttac ctcggaaaat tgaattcgac cagggatggc tgagaatcga
agacattgtc 120gacatcgcgg aagggtcggc tagggtagcg ttgtccgata
ctgctgcatt tcgctctggc 180attaagcgag gcgcggcctt cctggaccgt
ctgctgcacg aagagggcac gatctatggc 240gttactaccg gctacggcga
ttcgtgcacg gtgacggtgc cgcttgagct agtcgccgag 300ttgccgcgcc
agctctatat ctatcacggc tgtggactgg gcgagtattt gagtccggga
360cagacgcgcg ccgtgttggc aacccgcctc acatcgttgt gcaaagggtt
ttctggcgtg 420agcttagaac tgctggaaca aatcgtgagg ttactccagt
gcgatctatt gcctttgatt 480ccgtcggaag gctcggtggg tgccagcggc
gatctcactc cgctatcgta cctcgctgcg 540gttttgtgcg gcgagggcga
cgtttggcgt aacggcgtcc atgtaagcgc cgcgcaagca 600ctagccgagg
gcggtatcac accgctgcgt ttgaggccca aagaaggatt ggcgatcatg
660aatgggacag cagtcatgac cgctttggct tgccttgctt atgtgcgcgc
cgattatctc 720actcgactgg tgactcggat cactgcactg gcgtcgtttg
cgcttgacgg catgcgcacc 780atttcgatgc cacattgttc gcagtga
807102268PRTUnknownObtained from an environmental sample 102Met Leu
Phe Gly Ile Tyr Pro Arg Trp Thr His Leu Met His Pro Ala1 5 10 15Asp
Leu Lys Thr Ser Thr Leu Pro Arg Lys Ile Glu Phe Asp Gln Gly 20 25
30Trp Leu Arg Ile Glu Asp Ile Val Asp Ile Ala Glu Gly Ser Ala Arg
35 40 45Val Ala Leu Ser Asp Thr Ala Ala Phe Arg Ser Gly Ile Lys Arg
Gly 50 55 60Ala Ala Phe Leu Asp Arg Leu Leu His Glu Glu Gly Thr Ile
Tyr Gly65 70 75 80Val Thr Thr Gly Tyr Gly Asp Ser Cys Thr Val Thr
Val Pro Leu Glu 85 90 95Leu Val Ala Glu Leu Pro Arg Gln Leu Tyr Ile
Tyr His Gly Cys Gly 100 105 110Leu Gly Glu Tyr Leu Ser Pro Gly Gln
Thr Arg Ala Val Leu Ala Thr 115 120 125Arg Leu Thr Ser Leu Cys Lys
Gly Phe Ser Gly Val Ser Leu Glu Leu 130 135 140Leu Glu Gln Ile Val
Arg Leu Leu Gln Cys Asp Leu Leu Pro Leu Ile145 150 155 160Pro Ser
Glu Gly Ser Val Gly Ala Ser Gly Asp Leu Thr Pro Leu Ser 165 170
175Tyr Leu Ala Ala Val Leu Cys Gly Glu Gly Asp Val Trp Arg Asn Gly
180 185 190Val His Val Ser Ala Ala Gln Ala Leu Ala Glu Gly Gly Ile
Thr Pro 195 200 205Leu Arg Leu Arg Pro Lys Glu Gly Leu Ala Ile Met
Asn Gly Thr Ala 210 215 220Val Met Thr Ala Leu Ala Cys Leu Ala Tyr
Val Arg Ala Asp Tyr Leu225 230 235 240Thr Arg Leu Val Thr Arg Ile
Thr Ala Leu Ala Ser Phe Ala Leu Asp 245 250 255Gly Met Arg Thr Ile
Ser Met Pro His Cys Ser Gln 260 2651031641DNAUnknownObtained from
environmental sample 103atgatcacca tccggggaga aggactcacc atcgcccaga
tcgccgccgt cgcgcgcggg 60gcgccggtgc ggatgaccga tgacgaggcc gtgctggcgc
gcgtacgcgc cagccgccag 120cggatcgtcg aggcgctcga gtcgggccag
cagatctacg gcgtgaccac gctctacggc 180ggcatggccg acagggtcgt
cccggcggac cggctcgaag cgctgcagcg ggtgtcgctc 240tggcatcaca
aggtcggcgc cggcccccgc ctgccggtgc cggacgtgcg cgcggcgatg
300ctgctgcgtg ccaactcgct gttgaagggg gcctcgggcg tccgcgtgga
gctcgtcgag 360cgctacgtgg cgttcctgaa cgccggagcg acgccgcacg
tctatcagcg cggctcgatc 420ggggcgagcg gcgacctctc gccgctggcc
tacatcggtg cggccgtgat cggcctcgac 480cgggcctggc tcgtcgacct
tggcgaagag accctcgatt gcctgacggt gctgcgccgg 540ctcggcctcg
aaccgatgat cctgcagccg aaggagggcc tggcgctctg caacggcacc
600gccgcctcga ccggggtggc ggccaactgc atcgagcgcg ccttcgccct
gaccgcgctc 660gcactcggga cgcatgcgct ctacttccag gcgctcgacg
ccacgaccca gtcgttcgag 720cccttcgtgc atgcgctcaa gccccacccg
ggccaggtct ggaccgcccg ggagatggcc 780gcgctgctcg acggctccgg
cctcgtgcgc agcgaggccg ctggcgatcg tgcccaccgc 840cggggcggac
tgatccagga ccgctactcc ctgcgctgca tgccccagtt cgtcggtccc
900atcgtcgacg gcctggccga ggctgcccgc cagatcgaga tcgaagccaa
ttccgccaac 960gacaatccgc tgatcgatcc ggagaccggc gaggtgttcc
acaccggcaa cttcctggcg 1020gagtacaccg ccgtggcgat ggatcggctg
cgctaccacc tcggcatgct ggccaagcac 1080ctggacacgc agatcgcgct
gctggtggcg cccgagttca gccgcggcct gagcccgtcg 1140ctggcgggca
acctcgaacc ggggctcaac gtcggcctga agtcgctgca gatctcgtgc
1200aacagcctga tgccgctgct gacgttctat ggccagtcga tggccgaccg
cttcccgacg 1260catgcggagc agttcaacca gaacatcaac tcccaggcga
tgaacggtgc caatctggcc 1320cgcgactcgg tggaggtgct ggcccacttc
atggccaacg ccctcgtctt cgccgtacag 1380gccgtcgagc tgcgagcctg
gctggtggct ggcacctacg acgcatccga ggtgctctcg 1440ccggcgaccc
gcccgctgta tgcggccgcg cgcgccgccg ccgccggtcc gcccggaccg
1500caccgcccgc tcgtctggaa cgacaccgac ggctttctgg aggacaaggt
ccgcggcgtg 1560atgtcgagca tcctggaggg cggcgccgtg ccggaggccg
tcgcgccggt gcgcgagcgt 1620ctccgtgcgc atcgagggta g
1641104546PRTUnknownObtained from environmental sample 104Met Ile
Thr Ile Arg Gly Glu Gly Leu Thr Ile Ala Gln Ile Ala Ala1 5 10 15Val
Ala Arg Gly Ala Pro Val Arg Met Thr Asp Asp Glu Ala Val Leu 20 25
30Ala Arg Val Arg Ala Ser Arg Gln Arg Ile Val Glu Ala Leu Glu Ser
35 40 45Gly Gln Gln Ile Tyr Gly Val Thr Thr Leu Tyr Gly Gly Met Ala
Asp 50 55 60Arg Val Val Pro Ala Asp Arg Leu Glu Ala Leu Gln Arg Val
Ser Leu65 70 75 80Trp His His Lys Val Gly Ala Gly Pro Arg Leu Pro
Val Pro Asp Val 85 90 95Arg Ala Ala Met Leu Leu Arg Ala Asn Ser Leu
Leu Lys Gly Ala Ser 100 105 110Gly Val Arg Val Glu Leu Val Glu Arg
Tyr Val Ala Phe Leu Asn Ala 115 120 125Gly Ala Thr Pro His Val Tyr
Gln Arg Gly Ser Ile Gly Ala Ser Gly 130 135 140Asp Leu Ser Pro Leu
Ala Tyr Ile Gly Ala Ala Val Ile Gly Leu Asp145 150 155 160Arg Ala
Trp Leu Val Asp Leu Gly Glu Glu Thr Leu Asp Cys Leu Thr 165 170
175Val Leu Arg Arg Leu Gly Leu Glu Pro Met Ile Leu Gln Pro Lys Glu
180 185 190Gly Leu Ala Leu Cys Asn Gly Thr Ala Ala Ser Thr Gly Val
Ala Ala 195 200 205Asn Cys Ile Glu Arg Ala Phe Ala Leu Thr Ala Leu
Ala Leu Gly Thr 210 215 220His Ala Leu Tyr Phe Gln Ala Leu Asp Ala
Thr Thr Gln Ser Phe Glu225 230 235 240Pro Phe Val His Ala Leu Lys
Pro His Pro Gly Gln Val Trp Thr Ala 245 250 255Arg Glu Met Ala Ala
Leu Leu Asp Gly Ser Gly Leu Val Arg Ser Glu 260 265 270Ala Ala Gly
Asp Arg Ala His Arg Arg Gly Gly Leu Ile Gln Asp Arg 275 280 285Tyr
Ser Leu Arg Cys Met Pro Gln Phe Val Gly Pro Ile Val Asp Gly 290 295
300Leu Ala Glu Ala Ala Arg Gln Ile Glu Ile Glu Ala Asn Ser Ala
Asn305 310 315 320Asp Asn Pro Leu Ile Asp Pro Glu Thr Gly Glu Val
Phe His Thr Gly 325 330 335Asn Phe Leu Ala Glu Tyr Thr Ala Val Ala
Met Asp Arg Leu Arg Tyr 340 345 350His Leu Gly Met Leu Ala Lys His
Leu Asp Thr Gln Ile Ala Leu Leu 355 360 365Val Ala Pro Glu Phe Ser
Arg Gly Leu Ser Pro Ser Leu Ala Gly Asn 370 375 380Leu Glu Pro Gly
Leu Asn Val Gly Leu Lys Ser Leu Gln Ile Ser Cys385 390 395 400Asn
Ser Leu Met Pro Leu Leu Thr Phe Tyr Gly Gln Ser Met Ala Asp 405 410
415Arg Phe Pro Thr His Ala Glu Gln Phe Asn Gln Asn Ile Asn Ser Gln
420 425 430Ala Met Asn Gly Ala Asn Leu Ala Arg Asp Ser Val Glu Val
Leu Ala 435 440 445His Phe Met Ala Asn Ala Leu Val Phe Ala Val Gln
Ala Val Glu Leu 450 455 460Arg Ala Trp Leu Val Ala Gly Thr Tyr Asp
Ala Ser Glu Val Leu Ser465 470 475 480Pro Ala Thr Arg Pro Leu Tyr
Ala Ala Ala Arg Ala Ala Ala Ala Gly 485 490 495Pro Pro Gly Pro His
Arg Pro Leu Val Trp Asn Asp Thr Asp Gly Phe 500 505 510Leu Glu Asp
Lys Val Arg Gly Val Met Ser Ser Ile Leu Glu Gly Gly 515 520 525Ala
Val Pro Glu Ala Val Ala Pro Val Arg Glu Arg Leu Arg Ala His 530 535
540Arg Gly545
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